Virulence-encoding DNA sequences of Strepococcus suis and related products and methods

ABSTRACT

The invention provides DNA sequences which code for polypeptides which are characteristic for the virulence of the pathogenic bacterium Streptococcus suis and parts thereof, and polypeptides and antibodies derived therefrom. The sequences code for a polypeptide of 90,000-120,000 daltons or a polypeptide of higher molecular weight containing such a polypeptide, and for a polypeptide of 135,000-136,000 daltons (muramidase released protein), or parts thereof. The sequences themselves, and also the polypeptides and antibodies derived therefrom, are used for diagnosis of and protection against infection by S. suis in mammals, including man.

FIELD OF THE INVENTION

The invention is in the field of veterinary and human preventivemedicine, in particular that of the diagnosis of and protection againstinfection by pathogenic strains of the bacterium Streptococcus suis.

Infections with Streptococcus suis serotype 2 in young pigs at about thetime of weaning have been a growing problem in the Netherlands since1983. The disease is characterised by meningitis, arthritis, sepsis anddeath (Clifton-Hadley 1983, ref. 6; Vecht et al. 1985, ref. 44; Windsor1977, ref. 50). It is estimated that 5-10 per cent of farms haveproblems of this type. The mortality is estimated at 2.5% and themorbidity in affected farms is on average 2-5%. Therapeutic andpreventive measures have only a limited effect. The economic damage isaccordingly appreciable. The disease is a zoonosis. Humans are alsosusceptible to this infection, with the risk of sepsis and meningitiswith possibly permanent side-effects; rare cases of death have beenreported (Arends and Zanen 1988, ref. 2). This related mostly to casesof people with a skin wound coming into contact with infected pork. Inparticular, pig farmers and slaughterhouse staff belong to the riskgroup.

There are indications that the increased rate of illness on pig farms inthe Netherlands since 1983 is to be ascribed to the import of breedinganimals which are carriers of S. suis type 2. Carriers are often healthyadult pigs which harbour the streptococci in the tonsils and mucosa ofthe upper respiratory tract. The infection is transmitted via thesecarriers to susceptible animals, frequently piglets at weaning age.Diagnosis of animals which are already sick or have died is based onisolation and determination of S. suis type 2 from clinical samples ororgans after necropsy. Detection of carriers is based on bacteriologicalexamination of nose or throat swabs or tonsil biopsies using aselective/elective medium (Van Leengoed et al. 1987, ref. 27). On thebasis of diagnostic testing to detect carriers, it should be possible toset up a control programme. However, testing for carriers using theconventional becteriological techniques is time-consuming, whichcomplicates the processing of large numbers of samples; there is also arisk of false negative results due to overgrowth with contaminants.Finally, interpretation of the test demands a great deal of experience.Moreover, diagnosis and possible control on the basis of diagnosis arefurther complicated by the occurrence of differences in pathogenicitywithin the S. suis type 2 species. Regular testing for carriers within acontrol programme is sensible only if truly virulent strains of S. suistype 2 can be differentiated from avirulent strains. Current diagnostictechniques do not make such discrimination. Consequently, control basedon the detection of carriers of virulent S. suis type 2 strains is notyet possible.

Differences in virulence are ascribed, inter alia, to the presence orabsence of virulence factors. In 1984, Arends and Zanen (ref. 1)described "lysozyme-positive proteins" in human strains. In a study withexperimental animals it was found that a "lysozyme-positive" strain(D-282) was pathogenic for gnotobiotic pigs, in contrast to a"lysozyme-negative" strain (T-15) (Vecht et al. 1989, ref. 43). The"lysozyme-positive protein " is probably identical to themuramidase-released protein (MRP) of strain D-282.

The pig industry in the Netherlands and many other countries has apyramid structure, with a small number of breeding herds at the top,from where animals are distributed to replication herds. These supply alarge number of fattening herds, supplying animal products toslaughterhouses. A control program based on diagnosis (certification offarms, elimination of positive carriers, import requirements) shouldprimarily aim at creating herds which are free of S. suis type 2 high inthis pyramid. A vaccine would primarily be useful in affecting herdslower in the pyramid. Furthermore, means and methods for diagnosinginfections by Streptococcus suis in human medicine can be of value.

SUMMARY OF THE INVENTION

The object of the invention is to provide methods and means which makeit possible, in a more effective manner than hitherto, to detectinfections by Streptococcus suis on the one hand and to prevent suchinfections by elimination of infected pigs and carrier pigs on the otherhand.

This object is achieved by using a DNA sequence from the gene whichcodes for a virulence characteristic of S. suis. In this context, avirulence characteristic is defined as a polypeptide whose presence isassociated with the virulence of an organism, in this case the bacteriumS. suis, in particular serotype 2.

Two genes of virulent strains of S. suis type 2 have been found whichcode for two proteins, which are designated MRP (muramidase releasedprotein) and EF (extracellular factor) and which appear to becharacteristic for virulence (virulence factors). MRP and EF are highmolecular weight proteins. MRP (136kd) is a protein associated with thecell envelope and can be released from the cell wall by muramidase. EF(110kD) is an extracellular product which is secreted by the bacteriuminto the growth medium. EF has higher molecular weight counterpartswhich are denoted herein as EF* .

The invention provides new diagnostic methods which are able todifferentiate between virulent and avirulent strains. These methods arebased on the genes encoding MRP, EF and EF* and their expressionproducts. On the basis of the expression of one or both proteins by saidgenes, three different phenotypes of S. suis type 2 have been found todate: i.e. the MRP+ EF+ phenotype, the MRP+ EF- phenotype and the MRP-EF- phenotype. 77% (N=111) of strains isolated from organs of pigsshowing clinical symptoms of disease were found to possess the MRP+EF+phenotype, while 86% (n=42) of isolates from tonsils of non-suspectnormal slaughter pigs were found to possess the MRP- EF- phenotype. TheMRP+ EF- phenotype was most frequently found (74%) (n=27) in isolatesfrom human patients with infections of S. suis type 2 (see FIG. 10).Hence infected animals and carriers of virulent strains can be detected,and a vaccine based on MRP, EF and/or EF* can be developed. Using thediagnostic methods for detecting carriers and infected pig herds and/orusing vaccines based on MRP, EF and/or EF* , a program for controllinginfection by S. suis type 2 in pig herds can be developed.

The invention therefore also relates to the DNA sequence of the genewhich, apart from coding for specific high molecular weightpolypeptides, codes for the 90-120 kDa polypeptide which is acharacteristic of S. suis virulence, which gene, hereinafter designatedthe ef gene has the nucleotide sequence according to SEQ ID No: 1 for S.suis serotype D-282, and to equivalent sequences and to parts of saidsequences. The nucleotide sequence of the entire region coding for EFand the flanking sequences have been determined. Analysis of thesequence of the ef gene SEQ ID NO: 1provides an open reading frame of2529 nucleotides which codes for a polypeptide of 843 amino acids(calculated molecular weight 90,014). Monoclonal antibodies generatedagainst the 110 kDa EF protein recognised proteins with a highermolecular weight in culture supernatants of all 38 strains with aMRP+EF-phenotype. This indicates that certain of the 110 kDA EF and thehigh molecular weight proteins are identical. None of the 91 strainswith a MRP+ EF+ phenotype was found to produce these high molecularweight proteins. At the same time, DNA probes based on the gene whichcodes for the 110 kDa EF were found to hybridise with genes which codefor the high molecular weight proteins of MRP+ EF- strains. Thisindicates that the 110 kDa EF and the high molecular weight proteins arerelated, which implies that at least part of the ef gene, from strainswith a MRP+ EF- phenotype, is identical to the ef gene of strains withthe MRP+ EF+ phenotype. The higher molecular weight counterpart of theprotein EF is designated herein as EF* , and the gene encoding it as theef* gene. The corresponding nucleotide and amino acid sequences arerepresented in SEQ ID NO: 2

The invention also relates to the DNA sequence of the gene which codesfor the 135-136 kDa polypeptide which is also a virulence characteristicof S. suis, which gene, hereinafter designated the mrp gene, has thenucleotide sequence according to SEQ ID NO: 3 for S. suis serotype 2strain D-282, and to equivalent sequences and to parts of saidsequences. The nucleotide sequence of the entire region coding for MRPand the flanking sequences have been determined. Analysis of thesequence and the flanking sequences have been determined. Analysis ofthe sequence of the mrp gene SEQ ID NO: 3 shows an open reading frame of3768 nucleotides which codes for a polypeptide of 1256 amino acids(calculated molecular weight 135,794).

In this context, an equivalent sequence comprises a sequence which isessentially the same as the sequence shown but can display slightdifferences, such as point mutations, or other modifications which maybe caused by substitution, deletion, insertion or additional. Similarly,an equivalent sequence also comprises a sequence which, despite anydifferences in nucleotide sequence, hybridises with the sequence shownor with its complement, and also comprises a related sequence whichmeans that it codes for the same amino acid sequence despite differencesin nucleotide sequence.

The invention also relates to a recombinant polynucleotide whichcontains an ef/ef* gene and/or mrp gene sequence as described above, inthe presence of a regulating sequence. A recombinant of this type, suchas a virus vector, a plasmid or a bacterium, can be used for expressionof the gene or of relevant parts thereof in a desired environment, forexample for the production of immunogenic peptides intended for thediagnosis of an infection, or for controlling infection with virulentstrains of S. suis by vaccination.

Polynucleotide probes which contain a sequence as described above,derived from a gene which codes for a virulence characteristic of S.suis, also form part of the invention. A probe of this type inparticular corresponds with part of the nucleotide sequence of one ofthe two said genes. The probe can be used for direct detection of thepresence of sequences of virulent strains of S. suis. The probe can alsobe used as a basis for a primer for the multiplication ofpolynucleotides (for example in a polymerase chain reaction) as part ofa diagnostic method or a protection method.

A suitable polynucleotide probe was found to be a partial sequencecontaining at least 10 nucleotides, preferably at least 15 nucleotides,up to 835 nucleotides from the sequence 1100-1934 of the mrp gene.Another suitable polynucleotide probe was found to be a partial sequencecontaining 10-417, in particular 15-417 nucleotides from the sequence2890-3306 of the ef* gene. These probes differentiate effectivelybetween pathogenic and non-pathogenic strains of S. suis. A combinationof such an mrp based probe and an ef* based probe is an especiallypowerful diagnostic tool.

The invention also relates to polypeptides which are derived from apolynucleotide sequence described above. A polypeptide of this type iseither coded by said sequence or obtained by expression of said sequenceand essentially corresponds to a S. suis protein characteristic ofvirulence, or to a part thereof. A polypeptide of this type can, forexample, be used as an antigen in an immunoassay, as an immunogen in theimmunisation of mammals or as an immunogen for the production ofantibodies for diagnostic purposes. The antibodies generated in this wayalso form part of the invention. Such antibodies can be polyclonal ormonoclonal and can be conjugated with a marker (enzyme, isotope,luminescent substance or complex-forming agent); the antibody can alsobe bound to solid carriers or substrates.

The invention also relates to methods for the detection of an infectionby a pathogenic strain or by a non-pathogenic strain of S. suis, inwhich one or more polynucleotide probes, polypeptides and/or antibodiesas described above are used. "Infection" signifies here the presence ofthe pathogenic organism, both in the case where there are clinical signsof disease (infection in a narrow sense) and in the case where there areno clinical signs of disease (infection in a broad sense, ofcontamination). For immunoassays, such as a determination of thepresence of antigens of and/or antibodies against S. suis in a sample orin clinical material, it is possible, for example, to use on amicrotiter plate a polypeptide (110 kDa) which is encoded by the ef/ef*gene or a part thereof, and/or an antibody which has been generatedagainst such a polypeptide. In addition, it is also possible to use apolypeptide (136 kDa) encoded by the mrp gene or a part thereof, and/oran antibody which has been generated against such a polypeptide. Thediagnostic methods can be carried out using procedures known per se.Examples are Enzyme-Linked Immunosorbent Assays (ELISA) and DoubleAntibody Sandwich (DAS)-ELISA.

The methods described above can be carried out with the aid ofdiagnostic kits. A diagnostic kit according to the invention contains,respectively, at least one polynucleotide or a polypeptide whichcorresponds to or is derived from a sequence of the ef/ef* gene or mrpgene or a part thereof or contains an antibody which has been generatedagainst the polypeptide derived from one of the said ef/ef* and mrpsequences. It is also possible to use combinations of probes and thelike, in particular of ef* diagnostic agents and mrp diagnostic agents,or combinations of primers, for example for carrying out PCR. The kitscan also contain the components required for carrying out diagnoses,such as reagents (labelling substances, dyes and the like), supports(filters, plates and the like), media and calibrating agents as well asa manual for carrying out the diagnosis.

The invention also relates to a method for protecting mammals againstinfection by Streptococcus suis, in which method a polynucleotide, apolypeptide or an antibody as described above is used. When an antibodyis used, the method is a passive immunisation, that is to say there isdirect provision of antibodies against the pathogenic organism; sinceantibodies which are derived from EF, EF* and MRP are directed againstthe most virulent forms of S. suis, a procedure of this type can be aneffective method for protecting against, or controlling, infection,especially if the animal to be protected is not itself able to producesufficient antibodies, for example if infection has already taken placeor in the case of young animals.

Another form of passive immunisation in the case of pigs is theadministration of antibodies to the piglets via the colostrum from thesow. In this case the dam is actively immunised with one or bothpolypeptides during pregnancy, that is to say before the birth of thepiglets. When a polypeptide or a polynucleotide (optionally in the formof a recombinant organism) is used, the procedure is an activeimmunisation, the animal to be protected being stimulated, by means ofthe immunogenic polypeptide which is administered directly or in theform of a gene for expression, to produce antibodies.

Another suitable method of immunisation is the administration of apolypeptide from which the activity responsible for virulence has beenneutralised. Such a polypeptide should then no longer be pathogenic,while immunogenic characteristics are retained. It can be obtained, forexample, by expression of a gene which has been modified with respect tothe original ef/ef* or mrp gene, such as by means of deletion.

Vaccines for protecting mammals against an infection by S. suis, whichvaccines contain a polynucleotide, a polypeptide or an antibody asdescribed above, also form part of the invention.

A particular vaccine according to the invention is a vaccine whichcontains a S. suis material which does not or does not completely bringto expression at least one of the polypeptides corresponding to EF andMRP. This material can originate from or can be formed by a possiblelive strain which is not virulent or is less virulent.

The role of virulence factors which are involved in the pathogenesis ofS. suis type 2 has been studied in vivo by means ofgnotobiotic/germ-free piglets with S. suis type 2 strains defined inrespect of virulence factors (MRP and EF). The animal experiments weremonitored by means of haematological, bacteriological and(histo)pathological analytical techniques.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1

Nucleotide sequence of the ef gene and the adjacent sequences and the EFamino acid sequence derived therefrom. The presumed ribosome bindingsite, the -35 and -10 regions of the presumed promoters, and the regionswith complementary symmetry are marked. The possible cleaving site forsignal peptidase is between nucleotides 498-499.

SEQ ID NO: 2

Nucleotide sequence of the fragment encoding the S. suis type 2 ef* geneof strain 1890 and the deduced amino acid sequence of the EF* protein ofclass I. The putative ribosome binding site, the -35 and -10 regions ofthe putative promoter sequences, the repetitive regions R1-R11, and theputative termination signals are indicated. The region between thenucleotides 2859 and 5228 is absent in the gene encoding the 110 kDa EFprotein. The region between the nucleotides 3423 and 4456 is absent inthe genes encoding the call IV and class V EF* proteins.

SEQ ID NO: 3

Nucleotide sequence of the 4.6 kb EcoRI-HindIII fragment with the mrpgene of S. suis type 2 and the MRP amino acid sequence derivedtherefrom. The probable ribosome binding site, the -35 and -10 regionsof the presumed promoter sequences, the region of complementary symmetrybeyond the mrp gene, the repeating amino acid sequences and the envelopeanchor region are indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Restriction maps of ef containing fragments, subcloned into theplasmid vector pKUN19 (24). The open reading frames are boxed.Restriction sites: B:BamHI; Bg:BglII; E:EcoRI; KKpnI; N;NarI; P;PstI;S;SnaBI; Sa:SalI, Sp:SpeI.

FIG. 2: Schematic representation of the gene encoding the 110 kDa EFprotein and the flanking regions. EF is encoded by the open readingframe 1 (ORF1). The 3' end of ORF1 is overlapping with the 5' end ofORF2. ORF2 and ORF3 are separated by a TAA stop codon. Restriction sitesof interest are indicated.

FIG. 3: Schematic representation of the PstI-SnaBI fragment of the ef*genes of 5 different classes of the ef gene. The arrows indicate therepeated amino acid units. The lines indicate regions present in thedifferent strains. The gaps indicate the regions lacking in thedifferent strains.

FIGS. 4A-4B: Nucleotide sequences near the ends of the fragments lackingin the ef* genes of class IV and V (A) and in the ef gene (B). Theuppermost and middle sequences represent regions flanking the left andright ends of the lacking fragments. The bottom sequences show thejunctions as found in the class IV and V ef* genes (A) and in the efgene (B). Directly repeated sequences are shown in boxes. The boldnucleotides indicate the first bases of the translational triplets. Thenumbers refer to the nucleotide positions in the ef* gene of class I(SEQ ID NO: 2).

FIGS. 5A-5B: A. Restriction maps of the DNA inserts of putativeMRP-positive recombinant bacteriophages. The thick line indicates theDNA region which is present in all of these clones. Restriction sites:E:EcoRI; H:HindIII; X:XbaI; K:KpnI; S:SacI. B. Parts of the DNA insertssubcloned in the plasmid vector pKUN19 (24).

FIG. 6: Western blot analysis of proteins, encoded by recombinantplasmids and recombinant bacteriophages, which have been selected withmonoclonal antibodies against MRP. Lane 1: negative control; proteinsextracted from the cell wall of a MRP-negative strain of S. suis. Lane2: crude MRP preparation which contains proteins extracted from the cellwall of strain D282. Lane 3: PMR10-1. Lane 4: pMR7-2. Lane 5: pMR9-1.Lane 6: pMR9-2. Lane 7: pMR10-1. Lane 8: pMR10-2. Lane 9: lambda GEM11with control insert. Lane 10: lambda clone 7. Lane 11: lambda clone 9.Lane 12: lambda clone 10. Lane 13: lambda clone 11.

FIG. 7: Western blot of the protoplast supernatant (PPS), culturesupernatant (Cult. Sup.), and membrane vesicle (Membr.) fractions probedwith anti-MRP/EF rabbit K191 serum (diluted 1:500). The lanedesignations are numbered strain designations.

FIG. 8: Western blot of cell culture supernatants of selected S. suistype 2 strains probed with rabbit anti-MRP/EF serum (K191), anti-MRPserum, and anti-EF serum (1:500 diluted). The PAbs revealed three S.suis type 2 phenotypes: MRP+EF+, MRP+EF- and MRP- EF⁻. The lanedesignations are strain designations. Reference strain 1 (D-282) andstrains 3 to 9 (MRP+EF+) were isolated from pigs with S. suismeningitis. Reference strain 2 (T-15) and strains 10, 12, 16, and 17were isolated from the tonsils of healthy pigs. Strains 22, 23, 24, 25,26, 28, and 29 were isolated from human patients.

FIG. 9: Hydropathy profile (25) of MRP. Sequences above and below theline represent hydrophobic and hydrophilic regions respectively.

FIG. 10: Homology between the amino acid sequences at the C terminus ofMRP and several cell-envelope associated proteins of gram-positivebacteria. The amino acid sequence of S. suis MRP was compared with M6protein of Streptococcus pyogenes (20), protein A of Staphylococcusaureus (16), protein G of group G streptococci (10), AP4 of S. pyogenes(13), LP of Lactococcus lactis (46), WAP4 of S. mutans (11), T6 of S.pyogenes (38), and Fn-BP of S. aureus (39).

FIG. 11: Comparison of the amino acid sequence of the repeat units inMRP. Homologous regions are enclosed in boxes.

FIGS. 12A-12C: Fragments of the mrp and ef genes that were used as aprobe. On top of each figure is the localisation of restriction sitesthat were used to create the probes. The fragments which were used asprobes are indicated with solid bars. Left of the solid bar is theabbreviation of the probe. The arrow indicates the open reading frame(ORF) of each gene.

FIG. 12A: Probes of the mrp gene. The SacI and HindIII sites are notauthentic but are generated by subcloning fragments of the mrp gene.

FIG. 12B: Probes of the ef gene.

FIG. 12C: Probe of the ef* gene. The open bar indicates the insertsequence of ef* that is not part of the ef gene.

FIG. 13: Specificity of PCR. 10 ng of chromosomal DNA of S. suis type 2strains was used in the PCR with the primers p-15, p-16, p-34, and p-35.Lanes 1 to 4 contained amplified DNA of MRP⁺ EF⁺ strains (D282, 3, 10,and 22), lanes 5, 6, 7, and 9 of MRP⁺ EF* strains (17, 24, 26, 28),lanes 10 to 14 of MRP⁻ EF⁻ strains (T15, 12, 16, 18, and 25), and lane15 contained the negative control; all ingredients except DNA. Lanes 8and 16 contained 300 ng size marker Lambda DNA digested with HindIII andEcoRI.

FIG. 14: Dot spot hybridization of 13 S. suis type 2 strains with themrp and ef probes. In each experiment, row A contains 1 μg/spot DNA offour MRP⁺ EF⁺ strains; D282, 3, 10 and 22, and one positive control. RowB contains four MRP⁺ EF* strains: strain 17, 24, 26 and 28; and row Cfive MRP⁻ EF⁻ strains; T15, 12, 16, 18 and 25.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Cloning and nucleotide sequence analysis of the gene encoding the 110kDa extracellular protein of pathogenic Streptococcus suis type 2strains

MATERIAL AND METHODS

Bacterial strains and growth conditions. E. coli strains JM101 (29) andLE392 (33) were used as hosts for recombinant plasmids andbacteriophages. The pathogenic MRP⁺ EF⁺ strain D282 of S. suis type 2(43) was used for the isolation of chromosomal DNA. E. coli strains weregrown in Luria broth (30). Ampicillin was added as needed to a finalconcentration of 50 μg/ml. S. suis strains were grown in Todd-Hewittbroth (Oxoid, Ltd., London, England).

Construction and immunological screening of the DNA library. A DNAlibrary of S. suis type 2 strain D282 was constructed in LambdaGEM-11 asrecommended by the manufacturer of the cloning vector (Promega, Madison,USA). Recombinant bacteriophages were plated on E. coli strain LE392 andincubated for 16 h at 37° C.

Nitrocellulose filters (Schleicher and Schuell, Inc., Dassel, Germany)were placed on the plaques, and the plates were further incubated for 2h at 37° C. Recombinants that produced EF were visualized withmonoclonal antibodies (Mabs) directed against EF (Example 4). Boundantibodies were detected with anti-mouse serum conjugated with alkalinephosphatase (Zymed Laboratories, Inc., San Francisco, USA) as describedby Maniatis et al. (28). Selected EF positive clones were purified byseveral rounds of single plaque isolation and immunological screening.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)and Western blot analysis. Proteins were separated by SDS gelelectrophoresis in which 4% stacking and 6% separating gels were used(26). The separated proteins were transferred to nitrocellulose in aSemi-Dry transfer cell (Bio-Rad Laboratories, Richmond, USA). Specificproteins were visualized by use of polyclonal antibodies (Pabs, Example4) or Mabs directed against EF and anti-rabbit or anti-mouse seraconjugated with alkaline phosphatase (Zymed Laboratories).

DNA manipulations and nucleotide sequence analysis. Selected restrictionfragments were (sub)-cloned in the plasmid vector pKUN19 (24) bystandard molecular biological techniques (28). Progressiveunidirectional deletions were made with the Erase-a-Base system fromPromega (Madison, USA). DNA sequences were determined by the dideoxychain termination method (37). DNA and protein sequences were analysedby the software packages PCGENE (Intelli-genetics Corp., Mountain ViewCalif.) and Wisconsin GCG (University of Wisconsin).

RESULTS

Cloning of the ef gene. A DNA library was constructed by isolatingchromosomal DNA from strain D282 of S. suis type 2. This DNA waspartially digested with the restriction enzyme Sau3A and cloned into thebacteriophage LambdaGEM11 replacement vector. The library containedapproximately 5×10⁵ recombinants per μg of DNA. Two thousand plaques ofrecombinant phages were tested for the presence of antigenicdeterminants of EF by use of a Mab directed against EF. Two plaques werepositive. The expression of EF by the two selected recombinantbacteriophages was studied by Western blotting to analyse the proteinseluted from plaques. Both recombinants encoded a protein that comigratedwith EF secreted by S. suis and that was recognized by Mabs directedagainst EF. Thus both recombinant bacteriophages contained the completegenetic information for EF. The genetic information for EF on therecombinant bacteriophages was localized using restriction enzymeanalysis. The two clones shared a DNA region of about 13 kb. Parts ofthe common DNA region were subcloned into plasmid pKUN19 (FIG. 3) andthe proteins expressed by the recombinant plasmids were analyzed byWestern blotting. The plasmid containing the 6.8 kb KpnI-SalI fragment(pEF2-19, FIG. 3) encoded a protein with a molecular weight identical toEF, that was recognized by Mabs directed against EF. Plasmids containingthe 5.8 kb EcoRV-SalI or the 5.3 kb BglII-SalI fragment, however, didnot express EF. These data indicate that the EcoRV and the BglII sitesare within regions required for EF expression.

Nucleotide sequence of the ef gene. The nucleotide sequence of thefragment comprising the EF encoding region was determined. The sequenceSEQ ID NO: 1 showed the presence of 3 major open reading frames (ORFs).ORF1 (from nucleotide 361 to 2890), ORF2 (from nucleotide 2856 to 3459)and ORF3 (from nucleotide 3462 to 4053) encoded polypeptides of 843amino acids, of 201 amino acids and of 197 amino acids respectively.ORF1 contained a putative ATG start codon that is preceded by a sequencethat is similar to ribosome binding sites of several types ofgram-positive bacteria (17). In contrast, neither a start codon, nor aribosome binding site upstream of the ORFs 2 and 3 could be found. The3' end of ORF1 and the 5' end of ORF2 are overlapping, albeit indifferent frames. The ORFs 2 and 3 are separated by a single TAA stopcodon. Upstream of ORF1 two putative promoter sequences were found thatresembled the -35 and -10 consensus sequences of promoters commonlyfound in gram-positive bacteria (FIG. 1A). Downstream of ORF3, tworegions of extended dyad symmetry were present. Because both regionscontained a stretch of thymidine residues at the end of the potentialstem-loop structures, these potential transcription terminators arelikely to be rho-independent (34, 40). Because the sequence data did notreveal obvious transcription and translation signals upstream of, orwithin ORF2 and ORF3, it is doubtful that these ORFs express proteins.Another possibility is that the entire sequenced region contains onelarge open reading frame. This situation would occur if only twosequence errors were present: a +1 base pair frame shift in the region2856 to 2892 and an error in the stop codon at position 3459. Thispossibility was excluded by sequencing the ef gene from threeadditional, independently selected clones. Fragments of the initialclones were used as hybridization probes in order to isolate theseclones from the chromosome. The nucleotide sequences of these fragmentswere identical to those presented in SEQ ID NO: 1

Amino acid sequence of EF. Because only ORF1 was preceded by appropriateexpression/initiation signals, this ORF probably encodes EF. This wasconfirmed by subcloning two fragments into plasmid pKUN19: a SpeI-SnaBIfragment, that contained the entire ORFs 1 and 2 and a SpeI-NarIfragment, that contained ORF1 and the 5' end of ORF2 (FIG. 3). Theproteins expressed by the recombinant plasmids were analysed by Westernblotting. In E. coli both recombinant plasmids encoded a protein thatwas recognized by a Mab directed against EF and that had a molecularweight identical to that of EF secreted by S. suis. Therefore, ORF1encodes EF. The molecular weight of the ORF1 product calculated from thesequence (90,000) differed, however, from that of EF estimated from SDSpolyacrylamide gels (110,000).

EF is exclusively found in the supernatant of S. suis cultures, and thusthe protein is expected to be preceded by a signal peptide. Indeed, thefirst 46 amino acids of the deduced amino acid sequence of EF arecharacteristic of a typical signal peptide. An N-terminal part thatcontained six positively charged amino acids was followed by ahydro-phobic core of 21 amino acids and a putative signal peptidasecleavage site (45). The hydropathy pattern (25) of the deduced aminoacid sequence showed that, apart from the signal peptide, the EF proteinwas very hydrophilic and did not contain extended hydrophobic regions(cf. MRP, Example 3). No significant similarities were found between thededuced amino acid sequence of EF and the protein sequences in the EMBLData Library.

Although appropriate translation initiation signals upstream of ORF2 andORF3 could not be found, the deduced amino acid sequences of ORF2 andORF3 showed some properties which raised doubt to the idea that thoseframes are not expressed. The N-terminus of the putative ORF2 proteinshowed two highly repetative units of 57 amino acids (identity 82%). TheC-terminus of the putative ORF3 protein is functionally similar toC-terminal regions of several cell-envelope located proteins ofgrampositive bacteria (10, 12, 13, 16, 41). A hydrophobic region waspreceded by the conserved sequence Leu-Pro-X-Thr-Gly-Glu and followed bya highly hydrophilic region. This similarity suggests that the putativeORF3 protein is associated with the cell-envelope.

EXAMPLE 2

Cloning and nucleotide sequence analysis of genes encoding extracellularproteins of non-pathogenic Streptococcus suis type 2 strains

MATERIALS AND METHODS

Bacterial strains and growth conditions. Escherichia coli strain JM101(29) was used as host for recombinant plasmids. Seventeen MRP⁺ EF*strains of S. suis type 2 were isolated from human patients, fivestrains from tonsils of slaughthered pigs, seven strains from organs ofdiseased pigs and from two strain the origin was unknown (Example 4).The E. coli strain was grown in Luria broth (30). Ampicillin was addedas needed to a final concentration of 50 μg/ml. Streptococcus suisstrains were grown in Todd-Hewitt broth (Oxoid, Ltd., London, England).

Genomic DNA and oligonucleotides. Genomic DNA was isolated by lysis inproteinase K/SDS solution, extraction with phenol/chloroform andprecipitation with ethanol (28). The sequences of the oligonucleotidesused in the polymerase chain reaction (PCR) were:5'-ATGTAATTGAATTCTCTTTTTAAGT-3' and 5'-AAACGTCCGCAGACTTCTAGATTAAAAGC-3'.These oligonucleotides correspond to the positions 35 to 59 and 4308 to4279 in the S. suis type 2 ef gene SEQ ID NO: 1. The underlinedsequences indicate the recognition sites for the restriction enzymesEcoRI and XbaI.

DNA manipulations and nucleotide sequence analyses were carried out asdescribed in Example 1.

SDS - PAGE and Western blot analysis were carried out as described inExample 1.

Southern hybridization. DNA was transferred to Gene-Screen Plusmembranes (New England Nuclear Corp., Dreieich, Germany) as described byManiatis et al. (28). DNA probes were labeled with (³² P)dCTP (3000Ci/mMol, Amersham Corp., Arlington Heights, USA) by the use of a randomprimed labeling kit (Boehringer GmbH, Mannheim, Germany). The blots werehybridized with DNA probes as recommended by the supplier of theGene-Screen Plus membranes. After hybridization the membranes werewashed twice with a solution of 2×SSC (1×SSC is 0.15M NaCl plus 0.015Mtrisodium citrate, pH 7.0) for 5 min at room temperature and twice witha solution of 0.1×SSC plus 0.5% SDS for 30 min at 65° C.

Amplification of genomic DNA fragments by Polymerase Chain Reaction(PCR). PCR was used to amplify ef* sequences. Genomic DNA from differentMRP⁺ EF* strains of S. suis type 2 was used as a template. Amplified DNAfragments were isolated by agarose gelelectrophoresis and extractionfrom the gel with Gene Clean (Bio101, La Jolla, USA). The purifiedfragments were digested with EcoRI and XbaI and cloned into the plasmidpKUN19 (24). To exclude mistakes in the DNA sequences as a result of thePCR, six independently choosen clones were mixed prior to the nucleotidesequence analyses.

RESULTS

Western blot of EF* proteins. Culture supernatants of strains of S.suistype 2 belonging to the MRP⁺ EF* phenotype contained proteins that wererecognized by Mabs directed against EF (Examples 4, 6). The molecularweights (MW) of these proteins varied and were higher than that of EF.The proteins secreted by thirty-one strains of the MRP⁺ EF* phenotypewere compared with those secreted by a strain of the MRP⁺ EF⁺ phenotype.EF* proteins of five different molecular weight classes were found.Three strains synthesized an EF* protein of approximately 195 kDa (classI); eighteen an EF* of approximately 180 kDa (class II); one an EF* ofapproximately 175 kDa (class III); five an EF* of approximately 160 kDa(class IV) and four an EF* of approximately 155 kDa (class V).

Southern hybridization of ef* genes. The relationship between the genesencoding the 110 kD EF and the EF* proteins was studied. Chromosomal DNAof different MRP⁺ EF* strains (two representatives of each class weretaken) and of the MRP⁺ EF⁺ strain D282 (43) was digested with therestriction enzyme PstI. The various DNAs were hybridized with a ³² Plabeled EcoRV-SnaBI fragment containing the entire ef gene (FIG. 4, seeExample 1). The results showed that the DNA digests of the MRP⁺ EF* aswell as the MRP⁺ EF⁺ strains contained two PstI fragments that stronglyhybridized with the probe. These data indicated that the genes encodingthe 110 kDa EF and the EF* proteins are strongly related. The length ofthe largest hybridizing fragment was the same in all strains. Incontrast, the length of the smallest hybridizing fragment differedbetween the strains. Moreover, the variation in length of the smallesthybridizing fragment correlated well with the variation in the molecularweight of the EF* proteins secreted by the different strains. Since thesmallest hybridizing fragment is located at the 3' end of the ef gene(FIG. 4, Example 1), these data suggest that the ef and ef* genesdiffered mainly at their 3' ends.

Cloning of ef* genes. The genes encoding the different EF* proteins wereobtained using PCR to amplify the ef* containing DNA fragments. GenomicDNA of 5 different MRP⁺ EF* strains of S. suis type 2 (onerepresentative of each class) was used as a template. The amplifiedfragments were digested with restriction enzymes EcoRI and XbaI andcloned into E. coli.

Ef* gene of class I. The nucleotide sequence of a 6.8 kb EcoRI-XbaIfragment containing the entire ef* gene of class I and the regionsflanking it was determined. Analysis of the sequence revealed twoopen-reading frames (ORFs, SEQ ID NO. 2). The first ORF (from nucleotide361 to 5827) and the second ORF (from nucleotide 5830 to 6421) encodedpolypeptides of 1822 amino acids and 197 amino acids respectively. Basedon its size the first ORF is expected to encode the EF* protein (195kDa). The ORFs were separated by a single TAA stop codon. The first ORFcontained a putative ATG start codon that was preceded by a sequencesimilar to bacterial ribosome-binding sites (17). In contrast, thesecond ORF was not preceded by an appropriate start codon, nor by aputative ribosome-binding site.

The first 46 amino acids of the deduced amino acid sequence of the EF*protein had the characteristics of a typical signal peptide (45). The Cterminus of the mature part of the protein contained a number ofimperfect repeats of 76 amino acids. In the EF* protein of class I tenand a half repeats were present (denoted as R1 to R11, SEQ ID NO. 2. Thefirst four repeats were contiguous as were the last six and a halfrepeats. The fourth and the fifth repeated unit, however, were separatedby 113 amino acids and the fifth and the six unit by 22 amino acids(FIG. 5). The amino acid sequences of the last five and a half unit werehighly conserved, whereas the sequences of the first five units weremore variable. One particular amino acid sequence, Asn-Pro-Asn-Leu, wasconserved in all repeated units. No significant homology was foundbetween the EF* sequence of class I and any protein sequence in the EMBLData Library.

Ef* genes of class II, III, IV and V. Because the genes encoding thevarious EF* proteins differed mainly at their 3' ends, the nucleotidesequences of the small PstI fragments from the genes of class II, III,IV and V were determined. Comparison of the nucleotide sequences showedthat the various ef* genes were highly homologous in this region. Theef* genes differed, however, in the number and the arrangement ofrepeated units (FIG. 5). Unlike the ef* gene of class I, the ef* genesof class II and IV lacked the R9 and R10 regions; that of class IIIlacked the R6, R7 and R9 regions and that of class of IV lacked the R7,R8 and R9 regions. In addition, the ef* genes of class IV and V lacked afragment of 1,032 bp, which contained R4, R5 and parts of R3 and R6. Thetranslational reading frame of the region located at the 3' end of themissing fragment remained the same. The nucleotide sequences at theregions of the left and right ends of this 1,032 bp fragment showeddirect repeats of 9 bp (FIG. 6A).

Homology between ef* and ef genes. Because EF* proteins were recognizedby Mabs directed against the 110 kDa EF protein and because the ef*genes strongly hybridized with an ef-probe, the ef (Example 1) and ef*genes are assumed to be partly identical. Comparison of the nucleotidesequences of the ef and the ef* gene of class I showed that the 2,499nucleotides located at the 5' end of the ef and ef* encoding regionswere identical. Unlike the gene encoding the EF* protein of class I, thegene encoding the 110 kDa EF protein lacked a 2,368 bp fragment. As aresult of this deletion the reading frame was altered and the regionlocated at the 3' -end of the 2,368 bp fragment was translated indifferent frames in ef and ef* genes. Consequently, the 110 kDa EFprotein will not contain the repeated amino acid units. Analysis of thenucleotide sequences at the regions of the left and right ends of the2,368 bp fragment showed direct repeats of 10 bp (containing onemismatch) (FIG. 6B). Thus, the gene encoding the 110 kDa EF proteincould have been the result of a specific deletion of 2,368 bp within anef* gene. This would implicate that a S. suis strain that isnon-pathogenic can change into a strain that is pathogenic.

EXAMPLE 3

Cloning and nucleotide sequence of the gene encoding the 136 kDa surfaceprotein (MRP) of Streptococcus suis type 2

MATERIALS AND METHODS

Bacterial strains and growth conditions. Escherichia coli strain JM 101(supE,thi,(lac-proAB⁻)[F'traD36, lacI^(q) ZΔM15], 29) was used as a hostfor recombinant plasmid DNA. E. coli strain LE392 [F⁻ 'hsdR574(rk⁻'mk⁻), supE44, supF58, lacY1, or Δ(lac1ZY)6, galK2, galT22, mel1.trpR55](33) was used as a host for recombinant bacteriophages. Thepathogenic MRP⁺ EF⁺ strain D282 of S.suis type 2 (43) was used forisolating chromosomal DNA. E. coli strains were grown on LB broth (30).Solid LB medium contained 1.5% agar. Ampicillin was added as needed to afinal concentration of 50 μg/ml. streptococcus suis strains were grownin Todd-Hewitt broth (Oxoid Ltd.)

Southern hybridization was carried out as described in Example 2.

Construction and immunological screening of the DNA library were carriedout as described in Example 1 substituting MRP for EF.

SDS - PAGE and Western blot analysis were carried out as described inExample 1 substituting MRP for EF.

Nucleotide sequence analysis was carried out as described in Example 1.

RESULTS

Construction and screening of the library. Chromosomal DNA isolated fromstrain D282 of S. suis type 2 was partially digested with therestriction enzyme Sau3A. A DNA library was then constructed in thebacteriophage LambdaGEM11 replacement vector. Approximately 5×10⁵recombinants /μg DNA were obtained. A MAb directed against MRP was usedto screen 1,400 recombinant plaques for the presence of antigenicdeterminants of MRP. Five recombinant plaques reacted positive.

Characterization of the immunoreactive recombinants. The expression ofMRP by the five selected recombinant bacteriophages was studied byWestern blotting to analyse the proteins eluted from the plaques. Allfive recombinants encoded proteins that were recognized by MAbs directedagainst MRP. These proteins, however, had lower molecular weights (MW)than the MRP. Two clones encoded a protein of approximately 70 kDa(clones 10 and 11); two clones encoded a protein of approximately 80 kDa(clones 9 and 12), and one clone encoded a protein of approximately 90kDa (clone 7). Therefore, it was concluded that the five recombinantsdid not contain the complete genetic information for MRP. Restrictionenzyme analysis was used to compare the DNA inserts of the fiverecombinants. All clones shared a DNA region of about 17 kb (FIG. 7A).The DNA inserts differed, however, at the 3' and 5' ends. The variationin length at the 3' ends of the inserts correlated well with thevariation in MW of the truncated MRP proteins (cf. FIG. 7A). Thiscorrelation indicates that MRP encoding sequences were located at the 3'end of the DNA inserts. This was confirmed by subcloning fragmentsderived from the 3' end of the DNA inserts of clones 7, 9, and 10(FIG.7B) into plasmid vector pKUN19 (24). These construct encodedtruncated MRP proteins that were indistinguishable from the truncatedMRP proteins encoded by the recombinant phages (FIG. 8). Deletion of the0.7 kb EcoRI-KpnI fragment from these contructs stopped the expressionof the truncated MRP proteins. This suggests that the expression of mrpis initiated from the 0.7 kb EcoRI-KpnI fragment.

Cloning of the complete mrp gene. The complete gene for MRP was obtainedby hybridization of ³² P labeled KpnI-SacI fragment of pMR7-2(FIG. 7B)with EcoRI or KpnI digested chromosomal DNA of strain D282 of S. suistype 2. An EcoRi fragment of 7 kb and KpnI fragment of 7 kb hybridizedwith the probe. Because of its size, the EcoRi fragment was expected tocontain the complete mrp gene and because the expression of mrp isinitiated from the 0.7 kb EcoRI-KpnI fragment, the KpnI fragment wasexpected to contain only the 30' end of the gene. Fragments ranging from6 to 8 kb from EcoRI and KpnI digested chromosomal DNA were isolated ,and ligated into the EcoRI or KpnI site of pKUN19, whereafter theligation mixtures were transformed into E. coli JM101. Thirteen out of50 selected recombinants clones obtained with the KpnI fragmentshybridized with a MRP probe. All of these recombinant clones contained aplasmid (pMR-C) with a 7 kb KpnI insert. In contrast, of 2,500 selectedrecombinant clones obtained with EcoRI fragments, none hybridized withthe probe. Since the 7 kb EcoRI fragment is expected to contain thecomplete mrp gene, this finding indicates that expression of MRP istoxic in E. coli. Nevertheless, a plasmid (pMR11) with the entire mrpgene could be constructed by combining the 5' end of the mrp gene(isolated from pMR7-2) and the 3' end of the gene (isolated from pMR-C)by forced cloning. The copy number of this plasmid appeared to bestrongly reduced, about 20 times, compared to the copy number of pKUN19.The low copy number presumably reduced the toxic effects of high-levelexpression of MRP in E. coli to tolerable levels. The proteins producedby E. coli cells containing pMR11, were analysed by Western blotting. Asexpected, these cells produced a 136 kDa protein that comigrated withMRP and that was recognized by PAbs directed against MRP.

Nucleotide sequence of the mrp gene. The nucleotide sequence of a 4.6 kbEcoRI-HindIII fragment, containing the entire mrp gene and the regionsflanking it was determined. Analysis of the sequences, SEQ ID NO: 3,revealed an open reading frame of 3,768 nucleotides coding for apolypeptide of 1,256 amino acids (with a calculated MW of 135,794). Theputative ATG start codon is preceded by a sequence that is similar toribosome-biding sites in several types of gram-positive bacteria (17).The nucleotide sequence upstream of mrp resembles the -35 and -10consensus sequences of promoters commonly found in gram-positivebacteria. downstream of the mrp gene, a region showing extended dyadsymmetry can be detected. The potential hairpin structure in thecorresponding mRNA has a 12 bp stem separated by a 6 bp loop (ΔG =-15.9kcal/mol, calculated according to the rules of Tinoco et at., 40). Sincethe region of dyad symmetry is not followed by a thymidine-rich region,this potential transcription terminator signal appears to berho-dependent (34).

Amino acid sequence of MRP. MRP is a cell-envelope V2 associated pro andmust be translocated across the cytoplasmic membrane. The mature proteinmust therefore contain a signal peptide. Indeed, the first 47 aminoacids of the MRP have the characteristics of a typical signal peptide.An N-terminal part that contains seven positively charged residues isfollowed by a hydrophobic core of 21 amino acids and a putative signalpeptidase cleavage site (45, vertical arrow in SEQ ID NO: 3). Cleavageof the signal peptide would result in a mature protein with an MW of131.094, which is close to the MW (136 kDa) of MRP, estimated fromSDS-polyacrylamide gels (Example 4). A second hydrophobic region of 20amino acids was identified at the C terminus of the protein (FIG. 11).If this region is analogous to other envelope associated proteins ofgram-positive bacteria (10, 11, 12, 13, 16, 20, 38, 39, 46), it isprobably a cell membrane anchor. A short highly charged region and aregion with the Leu-Pro-X-Thr-Gly-Glu amino acid sequence, two regionsthat flank the presumed cellmembrane anchor, are also highly conservedamong surface proteins of gram-positive bacteria (FIG. 12). The aminoacid sequence Leu-Pro-X-Thr-Gly-Glu is putatively involved in cell-wallbinding.

Several other regions were identified in the MRP sequence. The matureform of MRP starts with a unique N-terminal sequence of 824 amino acids.This region is followed by a stretch of amino acids that is rich inproline residues: of 86 amino acids, 26 are proline residues. Thisregion is followed by three repeated units of 54 amino acids (FIG. 13).The first unit is separated from the second by 77 amino acids, but thesecond and third unit are contiguous. The sequences of the first and thesecond unit are highly conserved, whereas the third varies. The thirdrepeated unit is followed by the envelope anchor sequence. There waslittle homology between the MRP sequence and the protein sequences ofthe EMBL Data Library. One subsequence of MRP, amino acid residues619-985, however, shared some similarity (17.2% identity in a 377 aminoacids sequence) with a sequence of the fibronectin-binding protein ofStaphylococcus aureus (39).

EXAMPLE 4

Identification of two proteins associated with virulence ofStreptococcus suis type 2

MATERIAL AND METHODS

Streptococcal isolates. 180 strains of S. suis type 2 were obtained fromthree different sources. A total of 111 of these strains were obtainedfrom four Animal Health Services in the Netherlands. These strains wereisolated from organs of diseased pigs in the course of routinediagnostic procedures. Another 42 strains were isolated from tonsils ofhealthy pigs when they were slaughtered. 27 strains were isolated fromhuman patients with S. suis type 2 infections. Tonsillar and humanstrains were kindly provided by J. P. Arends, Streeklaboratorium voor deVolksgezondheid voor Groningen en Drente, Groningen, the Netherlands.All strains were typed as S. suis type 2 by using biochemical andserological methods, as described previously (44). Strain 1 (=D282) hadbeen determined previously to be virulent for newborn germfree pigs andproduced MRP, whereas strain 2 (=T-15) was nonvirulent and did notproduce MRP (43). Therefore, strains 1 (MRP⁺) and 2 (MRP⁻) were used asreference strains.

Culture conditions. A 1-day-old colony of each bacterial strain wasgrown on Columbia blood agar base (code CM 331; Oxoid, Ltd.) containing6% horse blood and was incubated overnight at 37° C. in Todd-Hewittbroth (code CM 189; Oxoid). Early stationary growth phase cultures wereobtained from the overnight cultures, diluted 10 times in Todd-Hewittbroth, and incubated for 4 h at 37° C.

Cell fractionation. Two cell fractions (protoplast supernatant andculture supernatant) were prepared from each of the 180 strains. Twomore cell fractions (protoplasts and membrane vesicles) were preparedfrom 23 strains selected randomly from the 180 strains. The 23 strainswere isolated from both diseased and healthy pigs, as well as from humanpatients. The four cell fractions were isolated from early stationarygrowth phase cultures in Todd-Hewitt broth. Protoplasts were isolated asdescribed by Van der Vossen et al. (47). After centrifugation in anEppendorf centrifuge, the protoplasts and the remaining supernatants(protoplast supernatant) were collected. Membrane vesicles were isolatedas described by Driessen et al. (9). The broth cultures were centrifugedat 4,000×g for 15 min, and the culture supernatants were collected.

Preparation of antigens and antisera. After a stationary growth phaseculture of strain D-282 was centrifuged, the supernatant was harvested,concentrated by filtration (type PM30 filters; Amicon Corp., Danvers,Mass.) to a concentration of 3 mg/ml, and dialysed once againstTris-buffered saline (50 mM, pH 7.5). This product was used as anantigen for raising polyclonal antibodies (PAb) in rabbits andmonoclonal antibodies (MAb) in mice. Rabbits were immunized byintramuscular and subcutaneous inoculation of 2 mg portions of proteinemulsified in equal volumes of Freund imcomplete adjuvant. Inoculationswere repeated the following day without the adjuvant. After 5 weeks therabbits were given intravenous booster inoculations of the same antigendose, but without the adjuvant. After 6 weeks, the rabbits wereexsanguinated. The serum of one rabbit (rabbit K191) was used as a probein the Western blot analysis.

MAbs against the protein EF were raised in BALB/c mice. The mice wereimmunized intraperitoneally with 0.5 ml portions of antigen containing25 μg of protein emulsified in equal volumes of Freund imcompleteadjuvant; 3 weeks later this procedure was repeated. After 5 weeks, themice were given intravenous booster inoculations of the same antigendose, but without the adjuvant. Hybridoma cell lines were prepared asdescribed by Van Zijderveld et al. (51). After 10 to 14 days, hybridomaswere tested for antibodies against EF by using an enzyme-linkedimmunosorbent assay. Hybridoma culture supernatants (diluted 1:2) werethen tested for anti-EF MAb on Western blots of culture supernatantsfrom strain D-282. Binding of MAb to the 110 kDa protein on thenitrocellulose filters was visualized with anti-mouse immunoglobulinsconjugated with alkaline phosphatase. The positive cells were clonedtwice by limiting dilution in microtiter plates. The resultingmonoclonal cell lines were used to produce ascites fluid inpristane-primed male BALB/c mice, as described previously (51).

Indirect enzyme-linked immunosorbent assay for screening hybridomaculture supernatants. Polystyrene microtiter plates (Greiner, Nurtingen,Germany) were coated for 16 h at 37° C. with a solution containing theconcentrated, dialysed culture supernatant from strain D-282 (see above)diluted in phosphate-buffered saline (pH 7.2; 0.075 mg of protein perml), and these preparations were incubated for 16 h at 37° C. Twofolddilutions of hybridoma culture supernatants were applied and tested asdescribed previously (51). Bound antibodies were incubated withanti-mouse immunoglobulins (diluted 1:500) that were conjugated withhorseradish peroxidase (HRPO, Nordic, Tilburg, The Netherlands).

Electrophoresis and Western blotting. The various cell fractions wereanalysed by SDS-PAGE as described by Laemmli (26) on 6 or 12%polyacrylamide. After electrophoresis, the proteins were stained withsilver (32). For Western blot analysis, the proteins were electroblottedonto nitrocellulose by using a Multiphor II Nova Blot system (PharmaciaLKB, Uppsala, Sweden). The blots were probed with a 1:500 dilution ofrabbit K191 PAb or with a 1:300 dilution of mouse MAb. Bound PAb werevisualized with anti-rabbit immunoglobulins conjugated with alkalinephosphatase. Bound MAb were visualized with a 1:1,000 dilution ofanti-mouse immunoglobulins conjugated with alkaline phosphatase (Zymed).

RESULTS

Protein profiles of four cell fractions of 23 selected strains. Theprotein profiles of the protoplast supernatants and membrane vesiclecell fractions from two S. suis isolates belonging to each group studied(diseased pigs, healthy pigs, and human patients), prepared from the 23strains examined were almost identical. In contrast, the proteinprofiles of the culture and protoplast supernatants differed distinctly.The protein profiles of isolates obtained from diseased pigs containedtwo protein bands that were absent in the protein profiles of mostisolates obtained from healthy pigs. One band represented a 136 kDaprotein, which was identified as MRP (43). In the SDS-PAGE analysis,separating gels containing 6% polyacrylamide revealed the presence ofMRP in both culture and protoplast supernatants (strains 1, 5, 24, and26). The second band represented a 110 kDa protein; because this proteinwas detected only in culture supernatants, it was designated EF. BothMRP and EF were present in the culture supernatant of virulent referencestrain 1 (=D-282), but were absent in all cell fractions of nonvirulentreference strain 2 (=T-15). The eight strains isolated from diseasedpigs contained both MRP and EF. Six of the eight strains isolated fromhealthy pigs lacked these proteins. Six of the seven strains isolatedfrom human patients contained MRP, but only three of the six alsocontained EF.

When rabbit K191 PAb directed against culture supernatants were used asprobes in the immunoblotting analysis, MRP and EF were clearly detectedin the cell fractions of S. suis type 2 strains. Protoplastsupernatants, culture supernatants, and membrane vesicles of strains 1,5, 24, and 26 contained the 136-kDa MRP (FIG. 9). Because MRP is a majorcomponent of protoplast supernatants, this protein must be localized inthe cell envelope of the bacteria. The culture supernatants of strains 1and 5 also contained the 110 kDa EF. Strains 24 and 26 contained MRP butnot EF; strains 2 and 13 contained neither of the proteins.

On the basis of the presence of MRP and EF in culture supernatants, thefollowing three phenotypes of S. suis type 2 strains were distinguished:MRP⁺ EF⁺, and MRP⁺ EF⁻, and MRP⁻ EF⁻ (FIG. 10). Proteins bands atvarious molecular masses higher than 150 kDa reacted with rabbit K191serum and were visualized in Western blots of culture supernatants ofstrains 17, 24, 25, 26, and 28. As such proteins were also recognized bythe anti-EF MAb, except in the culture supernatant of strain 25, the 110kDa EF was probably related to these proteins. Western blots probed withthe mouse anti-EF MAb showed that all of the strains with the MRP⁺ EF⁻phenotype contained higher molecular weight proteins in their culturesupernatants. However, none of the strains with the MRP⁺ EF⁺ phenotypecontained such proteins. Probing with rabbit K191 serum revealed highmolecular weight proteins in culture supernatants of 12 MRP⁻ EF⁻strains, including strain 25. Immunoblotting with anti-EF MAb showedthat these proteins were not related to EF. When the four cell fractionswere analysed by SDS-PAGE on 12% slab gels, no low molecular weightproteins associated with virulence were detected.

Protein profiles of culture and protoplast supernatants of 180 strains.All 180 S. suis type 2 strains were analysed for the occurrence of thethree phenotypes in culture and protoplast supernatants by using 6% slabgels. Eighty percent of the strains isolated from the organs of diseasedpigs had the MRP⁺ EF⁺ phenotype (Table 1).

                  TABLE 1                                                         ______________________________________                                        Prevalence of MRP and EF phenotypes in 180 streptococcal                      strains isolated from diseased pigs, from healthy pigs when                   they were slaughtered, and from human patients.                                        No. (%) of strains isolated from:                                    S. suis type 2                                                                           Organs of   Tonsils of  Human                                      phenotype  diseased pigs                                                                             healthy pigs                                                                              patients                                   ______________________________________                                        MRP.sup.+ EF.sup.+                                                                       86 (77)      1 (2)       4 (15)                                    MRP.sup.+ EF.sup.-                                                                       13 (12)      5 (12)     20 (74)                                    MRP.sup.- EF.sup.-                                                                       12 (11)     36 (86)      3 (11)                                    ______________________________________                                    

In contrast, only 2% of the strains isolated from tonsils of healthypigs had this phenotype; 86% of these strains were MRP⁻ EF⁻. Only 15% ofthe strains isolated from human patients had the MRP⁺ EF⁺ phenotype.Among the S. suis type 2 strains tested, far more human strains (74%)than porcine strains (12%) had the MRP⁺ EF⁻ phenotype; 89% of the humanstrains were MRP⁺. The MRP⁻ EF⁺ phenotype was not detected.

EXAMPLE 5

Virulence of Streptococcus suis type 2 strains in new-born germ-freepigs.

MATERIALS AND METHODS

Pigs. Fifty-two germ-free pigs, cross-breeds of Great Yorkshire andDutch Landrace, were obtained from four sows by caesarian sections. Sowsin both experiments were full sisters. Pigs were allotted to 12 groupseach consisting of 4 or 5 pigs. Each group was housed in a sterilestainless steel incubator. Housing and feeding were as described before(43).

Inocula. Ten S. suis type 2 strains belonging to either phenotypeMRP+EF+, MRP+EF-, or MRP-EF- were obtained from three sources: from apig with meningitis, from healthy pigs at slaughter, and from humanpatients (Table 2). The strains were biochemically and serologicallytyped as described earlier (44). Strains were stored as stocksuspensions on glass beads in Nutrient Broth with 15% glycerol at -70°C. A one-day-old colony of each strain, grown on Columbia blood agarbase (Code CM 331, Oxoid) containing 6% horse blood, was incubatedovernight at 37° C. in Todd-Hewitt broth (Code CM 189, Oxoid). Earlystationary growth phase cultures were obtained by diluting the overnightcultures in Todd-Hewitt broth (1:10) and incubated them at 37° C.Incubation was stopped after approximately 4 h, when the optical densityat 600 nm was 0.5. Cultures containing approximately 1 to 3×10⁹ CFU/mlwere then centrifuged at 4000× g for 15 min. The supernatant wasanalysed for MRP and EF. Then the pellets were washed and suspend at anA₆₀₀ =1 in a solution of phosphate-buffered saline (PBS), 139.89 mMNaCl, 2.68 mM KCl, 8.1 mM Na₂ HPO₄, 2.79mM KH₂ PO₄, ph 7.2, and thenused as inoculum. Bordetella bronchiseptica strain 92932, isolated fromthe nose of a pig with atrophic rhinitis, was used to predispose pigs toS. suis infection (23, 43). The strain was kept on Dorset egg medium.The inoculum was prepared by culturing a 48 hour old colony from sheepblood agar in brain heart infusion broth. After 18 h of incubation at37° C., this medium contained approximately 10⁹ CFU/ml. The brain heartinfusion broth was diluted (1:100) in PBS to prepare the inoculum.

Electrophoresis and Western blotting. The MRP/EF phenotypes of the S.suis strains used as inocula and of the isolates recovered at the end ofthe experiments were determined. SDS-PAGE as described by Laemmli (26)(6% polyacrylamide) and Western blotting were used to analyse cellculture supernatants of isolates recovered from nasopharynx of all pigs,and from inflamed tissue such as meninges or joints of affected pigs.After electrophoresis the proteins were stained with silver (32). ForWestern blot analysis, the proteins were electroblotted ontonitrocellulose by the Multiphor II Nova Blot system, according to therecommendations of the manufacturer (Pharmacia LKB). Nitrocellulosefilters were incubated either with a 1:1 mixture of mouse anti-MRPmonoclonal antibodies (MAb) (11.3 mg/ml) and anti-EF MAb (8.4 mg/ml)each in a 1:200 dilution, or with a 1:500 dilution of polyclonalanti-MRP/EF rabbit serum (K191) (8.2 mg/ml) (Examples 4, 6). Filterswere incubated with a 1:1000 dilution of anti-mouse immunoglobulinsconjugated with alkaline phosphatase (AP) or a 1:3000 dilution of APconjugated anti-rabbit immunoglobulin g(γ+κ) (Zymed). Bound antibodieswere visualized by adding the substrate bromochloroindolyl phosphate(Sigma, St. Louis, Mo.--nitro blue tetrazolium (Merck, Darmstad,Germany) in phosphatase buffer (100 mM NaCl, 5 mM MgCl₂, 100 mMdiethanolamine; ph 9.5).

Experimental design. The study consisted of two experiments with aninterval of five months. Five day old germ-free pigs were inoculatedintranasally with a plastic disposable syringe filled with a suspensionof B. bronchiseptica strain 92932 in brain heart infusion broth. Theinocula contained 0.84×10⁷ CFU in experiment I and 1.0×10⁷ CFU inexperiment II. Two days post inoculation (pi) the pigs were similarlyinoculated inside the sterile incubator with one of the ten S. suis type2 strains (Table 2).

The mean (±SD) inoculum size of these strains was 1.4 (+0.60)×10⁶ CFU.All inoculations consisted of a 0.5 ml bacterial suspension in eachnostril during the inspiratory phase of breathing. In both experimentsstrain 3 (MRP+EF+) was used as positive control and strain 12 (MRP-EF-)was used as negative control (see Results section). Pigs were killedeither when they became mortally ill or at the end of the experiment (3to 4 weeks pi), and they were subsequently necropsied.

                  TABLE 2                                                         ______________________________________                                        Experimental design.                                                          S. Suis          Source.sup.1 of                                                                           Dosage.sup.2                                                                          No. of                                   strain                                                                               S. suis   S. suis     of S. suis                                                                            pigs                                     no.   phenotype  isolation   inoculation                                                                           inoculated                               ______________________________________                                        3     MRP+EF+    meninges pig                                                                              1.84    5                                        3     MRP+EF+    meninges pig                                                                              1.96    4                                        10    MRP+EF+    tonsil pig  1.52    5                                        22    MRP+EF+    human       2.93    4                                        17    MRP+EF-    tonsil pig  1.26    4                                        24    MRP+EF-    human       1.22    4                                        28    MRP+EF-    human       1.23    4                                        12    MRP-EF-    tonsil pig  1.05    5                                        12    MRP-EF-    tonsil pig  0.98    4                                        16    MRP-EF-    tonsil pig  0.70    4                                        18    MRP-EF-    tonsil pig  1.10    4                                        25    MRP-EF-    human       0.97    4                                        ______________________________________                                         .sup.1 Strain 3 was isolated during routine diagnostic procedures from a      pig with meningitis. Strains 10, 12, 16, and 18 were isolated at slaughte     from the tonsils of healthy pigs. Strains 22 (no. 830544), 24 (no.            740113), 25 (no. 821021) and 28 (no 760366) were isolated from human          patients with S. suis type 2 meningitis. (Numbers between parentheses         refer to those by J. P. Arends and H. C. Zanen (2)).                          .sup.2 × 10.sup.6 CFU.                                             

Disease monitoring . Pigs were monitored daily for clinical signs ofdisease, such as fever, dysfunction of the CNS and lameness. Bloodsamples from each pig were collected three times weekly by venipunctureof the cranial vena cava. White blood cells were counted with aconducting counter (Contraves A.G., Zurich, Switerland) (18 ). Thenumber of neutrophils was calculated after differential count ofGiemsa-stained blood smears. Swabs specimens of nasopharynx and feceswere collected daily and plated directly onto Columbia agar containing6% horse blood. The presence of S. suis type 2 and of B. bronchisepticawas confirmed by slide agglutination test in which a suspension of themonocultures was mixed with the appropiate hyperimmune rabbit serum(DLO-Central Veterinary Institute, Lelystad, NL). After pigs werekilled, they were examined for pathologic changes. Tissue specimens ofthe CNS, serosae, liver, spleen, and tonsils were bacteriologically andhistologically examined as described before (43).

RESULTS

Electrophoresis and Western blotting. When immunoblots were used toanalyse culture supernatants of the S. suis strains before inoculation,three phenotypes were distinguish. Strains 3, 10, and 22 belonged to theMRP+EF+ phenotype, strains 17, 24, and 28 were of the MRP+EF- phenotype,and strains 12, 16, 18, and 25 belonged to the MRP-EF- phenotype. Therabbit polyclonal antibodies (PAb) recognized proteins that were greaterthan 150 kDa in the culture supernatants of the MRP+EF - strains. Thesehigh molecular weight proteins were also detected by the anti-EF MAb,indicating that the 110 kDa EF and the >150 kDa proteins share epitopes.In both the SDS-PAGE and Western blot, the phenotypes of the S. suisstrains used as inocula were identical to the phenotypes of the isolatescollected at the end of both experiments from tonsils and inflamedtissues of infected pigs.

Clinical signs of disease. In both experiments, rectal temperatures ofall pigs inoculated with strains of the MRP+EF+ phenotype increased fromday 2 pi onwards, with peaks at 41.8° C. between days 4 and 9. Rectaltemperatures of ten pigs inoculated with strains of the MRP+EF-phenotype were higher than 40° C. for short periods of 24 to 96 hbetween days 2 and 22. Frequency of fever was highest in the MRP+EF+groups (40%) (Table 3). The frequency of increased polmorphousleucocytes (PML) in blood was highest in the MRP+EF+ groups (Table 3).Analysis of variance was performed on the log of PML counts in bloodsamples of pigs inoculated with strains of the three phenotypes. Threedays before inoculations no significant differences were found betweenthe geometric mean PML counts of the three groups. From day one pionwards, the means of numbers of PML in blood samples of pigs inoculatedwith strains of the MRP+EF+ phenotype were significantly higher (p<0.01)then in either the MRP+EF+ groups or the MRP-EF- groups. On day 20 pi,the means in the MRP+EF+ and MRP+EF- groups did not differ significantlyfrom each other, but those means differed significantly (p<0.01) fromthe means in the MRP-EF- groups. Morbidity in pigs inoculated withstrains of the MRP+EF+ phenotype was 100%. From day 2 onwards,non-specific signs of systemic disease, such as depression, recumbency,lack of appetite, and fever were observed. During the following days,pigs showed more specific signs of disease, such as ataxia, circularmovements, opisthotonus, recumbency with paddling, and lameness. Thefrequency of specific signs of diseases in the MRP+EF+ groups was 57%(Table 3). Nine pigs died in the course of the experiment, and threewere killed in the terminal stages of disease. The mortality rate inthese groups was thus 12/18 (67). Nine pigs inoculated with strains ofthe MRP+EF- phenotype developed fever or granulocytosis or showed othernonspecific signs of disease, but did not show specific clinical signs,such as nervous disorders or lameness. Pigs in the MRP-EF- groups didnot develop clinical signs of disease (Table 3)

                  TABLE 3                                                         ______________________________________                                        Frequency of three parameters of disease observed in pigs                     inoculated with S. suis type 2 (10 strains belonging to three                 phenotypes)                                                                          Frequency.sup.1 (%) of 3 parameters of disease                                            PML                                                        S. suis  Fever     in blood Clinical signs of disease                         phenotype                                                                              >40° C.                                                                          >10.sup.10 /L                                                                          specific.sup.2                                                                        non-specific.sup.3                        ______________________________________                                        MRP+EF+  40        78       57      21                                        MRP+EF-  5         16       0       5                                         MRP-EF-  0         3        0       0                                         ______________________________________                                         .sup.1 Number of positive records/total number of records                     .sup.2 Lameness and nervous disorders such as ataxia, circular movements,     opisthotonus, and recumbency with paddling.                                   .sup.3 Depression, lack of appetite, and recumbency.                     

Pathologic findings are summarized in Table 4. Severe and frequentinflammations of the CNS, serosa, and joints were only detected in pigsinoculated with strains of the MRP+EF+ phenotype. Pneumonia andbronchitis were observed in various forms. Follicle formation in B cellareas and blast cell formation in T cell areas of the white pulp of thespleen--signs of active immune response--were more frequently observedin pigs inoculated with strains of the MRP+EF- phenotype (50%) than inpigs inoculated with strains of the MRP-EF- phenotype (22%) or strainsof the MRP+EF+ phenotype (11%) (Table 4). Some pigs inoculated withMRP+Ef+ showed lymphocytolysis in the germinal centres, while themarginal zone surrounding the white pulp was inflamed, signs of acuteseptichaemia in young animals (42). Active follicles in tonsils werealso more often seen in pigs inoculated with strains of the MRP+EF- orMRP-Ef- phenotype.

                  TABLE 4                                                         ______________________________________                                        Pathologic lesions detected in various tissues of pigs                        inoculated with S. suis type 2 (10 strains of three                           phenotypes                                                                              No. of pigs with pathologic lesions                                             phenotype  phenotype  phenotype                                   Tissue and  MRP+EF+    MRP+EF-    MRP-EF-                                     pathologic  (no.       (no.       (no.                                        lesions     tested = 18)                                                                             tested = 12)                                                                             tested = 22)                                ______________________________________                                        CNS                                                                           Meningitis.sup.1                                                                          12         0          0                                           Encephalitis.sup.1                                                                        10         1          0                                           Choroiditis 7          0          0                                           Malacia     5          0          0                                           Serosae/joints                                                                Peri-/epicarditis                                                                         11         1          1                                           Pleuritis   5          1          0                                           Peritonitis 14         6          0                                           Polyarthritis.sup.2                                                                       15         0          0                                           Lungs                                                                         Cath.       1          1          1                                           broncho-pneumonia                                                             Fibrinous   3          0          0                                           pneumonia                                                                     Interstitial                                                                              7          5          5                                           pneumonia                                                                     Bronchitis/ 2          2          3                                           Peribronchiolitis                                                             Liver                                                                         Periportal and/or                                                                         11         8          3                                           intralobular foci                                                             Spleen                                                                        Active white pulp                                                                         2          6          5                                           Active red pulp                                                                           4          0          2                                           Tonsil                                                                        Active follicles                                                                          3          9          12                                          Exudation in crypts                                                                       1          5          6                                           ______________________________________                                         .sup.1 Affecting cerebrum, cerebellum, pons, mesencephalon and medulla        oblongata in various combinations.                                            .sup.2 Affecting carpal, metacarpal, tarsal, metarsal, knee, elbow,           shoulder and hip joints in various combinations.                         

Bacteriologic findings. From day 1 one pi to the end of the experiment,the streptococcal strains and B. bronchiseptica were isolated daily fromnaso-pharyngeal and fecal swab specimens of all pigs. A Bacillus specieswas also isolated from day six pi onwards from pigs inoculated withstrain 16 (experiment I) and from day 19 pi onwards from pigs inoculatedwith strain 24 (experiment II). Pigs in the other groups remained freefrom contaminating bacteria.

At necropsy, S. suis type 2 was mostly isolated from organs and tissues(CNS, serosae, and joints) that also showed pathologic changes (Table5). B. bronchiseptica was only isolated from lungs and tonsils. Both S.suis and B. bronchiseptica were also isolated from the tonsils of allpigs.

                  TABLE 5                                                         ______________________________________                                        Isolation of streptococci from various tissues of pigs                        inoculated with S. suis type 2 (10 strains of three                           phenotypes).                                                                  No. of pigs from which S. suis                                                was isolated at necropsy                                                              phenotype   phenotye    phenotype                                             MRP+EF+     MRP+EF-     MRP-EF-                                               (no.        (no.        (no.                                          Tissue  tested = 18)                                                                              tested = 12)                                                                              tested = 22)                                  ______________________________________                                        CNS     14          0           0                                             Serosae  9          2           0                                             Joints  13          2           0                                             Lungs    6 (9)      0 (2)       2 (8)                                         ______________________________________                                         .sup.1 Numbers in parentheses indicate number of pigs from which B.           bronchiseptica was also isolated.                                        

EXAMPLE 6

Discrimination between Virulent and Nonvirulent Streptococcus suis type2 Strains by Enzyme-Linked Immunosorbent Assay

MATERIALS AND METHODS

Bacteria. 179 strains of S. suis type 2 obtained from three sources wereexamined: from organs of diseased pigs in the course of routinediagnostic procedures, from tonsils of healthy pigs at slaughter, andfrom human patients suffering from S. suis type 2 infection. SDS-PAGEand Western blotting techniques were used in an earlier study to detectMRP and EF in culture supernatants, and on the basis of these resultsstrains were categorized into three phenotypes: MRP+EF+, MRP+EF-, andMRP-EF- (Example 4). Also tested were 22 strains of S. suis serotypes 1to 22 (15), 22 other streptococci, 20 bacterial strains of 15 differentspecies, and one yeast (DLO-Central Veterinary Institute, Lelystad)(Table 6).

                  TABLE 6                                                         ______________________________________                                        List of microorganisms                                                        Group Microorganisms     Microorganisms                                       ______________________________________                                        A     Streptococcus pyogenes humanis                                                                   Other bacterial species:                             B     Streptococcus agalactiae                                                                         Staphylococcus aureus                                C     Streptococcus equi Staphylococcus                                                                epidermidis                                                Streptococcus equisimilis porcine                                                                Staphylococcus hyicus                                      Streptococcus dysgalactiae                                                                       Aerococcus viridans                                        Streptococcus zooepidemicus                                                                      Actinomyces pyogenes                                 D     Enterococcus faecalis                                                                            Escherichia coli (3x)                                      Enterococcus faecium                                                                             Klebsiella oxytoca                                         Enterococcus liquefaciens                                                                        Klebsiella pneumoniae                                      Streptococcus bovis (2x)                                                                         Micrococcus strain 3551                                    Streptococcus zymogenes                                                                          Micrococcus Luteus                                   E     Streptococcus group E                                                                            Pasteurella multocida                                G     Streptococcus group G (2X)                                                                       (4x)                                                 L     Streptococcus group L (2X)                                                                       Proteus vulgaris                                     p     Streptococcus group P                                                                            Salmonella typhimurium                               Q     Streptococcus group Q                                                                            Serratia liquefaciens                                      Streptococcus milleri III                                                                        Yeast:                                                     Streptococcus sanguis                                                                            Cryptococcus laurentii                                     Streptococcus uberis                                                    ______________________________________                                    

Culture condition and antigen preparation. A 1 day old colony of thebacteria grown overnight on Columbia blood agar base (code CM 331. OxoidLtd.) containing 6% horse blood was inoculated into Todd-Hewitt broth(code CM 189, Oxoid). After overnight growth at 37° C., cultures werecentrifuged at 4000× g for 15 min. At 600 nm the optical densities ofthe 20 hour cultures were found to vary from 0.60 to 1.04. Some specieshad lower densities, these were Bordetella bronchiseptica (0.23).Micrococcus species (0.08 to 0.15). Streptoccoccus equinus (0.36).Cryptococcus neoformans (0.05). Twofold serial dilutions of untreatedculture supernatants were used as test samples in the two DAS-ELISAs.Culture supernatant of S. suis type 2 strain D₂₈₂ (MRP+EF) wasconcentrated and partially purified by ultrafiltration (type PM30filters, Amicon Cooperation). It was diluted in phosphate-bufferedsaline (PBS) (136.89 mM NaCl. 2.68 mM KCl. 8.1 mM Na₂ HPO₄. 2.79 mM KH₂PO₄. pH 7.2), to a final protein concentration of 75 μg/ml. This productwas used as coating antigen for the selection of different monoclonalsin the direct competition ELISA and for screening hybridoma culturesupernatants in the indirect ELISA.

Preparation of polyclonal and monoclonal antibodies. Rabbit (Ra)polyclonal antibodies (PAb) directed against MRP and EF (Ra K₁₉₁) andthree different MAbs directed against EF were prepared as described inExample 4. MAbs that specifically recognize MRP were preparedessentially the same as MAbs that recognize EF. Antigen production andimmunization procedures in female BALB/c mice have been described(Example 4). Hybridoma cell lines were prepared as described (52). After10 to 14 days, hybridoma culture supernatants were tested for antibodiesagainst MRP in an indirect ELISA (see below). Hybridoma culturesupernatants (diluted 1:2) were then tested on Western blots of culturesupernatants of strain D-282 for antibodies directed against MRP. BoundMAb to the 136 kDa protein were visualized by using anti-mouseimmunoglobulins conjugated to alkaline phosphatase and the substratedescribed below. Five supernatants were found positive, and the cellsfrom these wells were cloned twice by limiting dilution in microtiterplates.

The five cell lines that were positive for anti-MRP antibodies and thethree cell lines that were positive for anti-EF antibodies were used toproduce ascites fluid in pristane-primed male BALB/c mice. MAbs directedagainst MRP and EF were purified from ascites fluid by ammonium sulphateprecipitation (50% saturation) and dialysed against PBS. The fiveanti-MRP MAbs were designated: MRP₁ to MRP₅, the three anti-EF MABs weredesignated: EF₁ to EF₃. The immunoglobulin isotype of all MAbs was IgG₁and was determined by double immunodiffusion with mouse isotype-specificantisera (Nordic) in gels of 1% agarose in PBS. The PAbs and MAbs werestored at -20° C.

Indirect ELISA for screening hybridoma culture supernatants. Polystyrenemicrotiter plates (Greiner, Nurtingen, Germany) were coated for 16 h at37° C. with the solution of concentrated and dialysed culturesupernatant of strain D-282 (see above). They were then diluted in PBS,pH 7.2 (75 μg/ml protein). Twofold dilutions of hybridoma culturesupernatants were added to the wells according to the proceduredescribed by Van Zijderveld et al. (51). After the plates were washed,antimouse immunoglobulins (diluted 1:500) conjugated with horse radishperoxidase (HRPO, Nordic) were added. After incubation for 1 h at 37° C.and five washings, the bound HRPO-antibody was then detected by theaddition of substrate, 0.1% (w/v) solution of recrystallized5-aminosalicylic acid (5-AS) (Merck) in 0.01M phosphate buffer, pH 5.95,containing 0.01M sodium EDTA to which H₂ O₂ had been added, immediatelybefore use to an end concentration of 0.005% (wt/vol). After 2 hincubation at room temperature, the absorbance was measured at 450 nmwith a Titertek Multiskan photometer (Flow Labs).

Direct competition ELISA. MAbs were selected with the direct competitionELISA and were used to develop the MRP and EF double antibody sandwich(DAS) ELISAs. Purified anti-MRP and anti-EF MAbs and rabbit PAbs wereconjugated to HRPO (Boehringer Mannheim, Germany) with the periodatemethod of Wilson and Nakane (49). Conjugated immunoglobulins were storedat -20° C. in 50% glycerol. Conjugate solutions were made in PBS-Twcontaining 5% fetal calf serum and 0.5% sodium chloride. 50 μl ofnonconjugated anti-MRP MAbs in serial twofold dilutions (range 1:20 to1:10,240) were added to the wells of polystyrene microtiter ELISA plates(Greiner) that had been coated with the culture supernatant of strainD₂₈₂ that had been partially purified in PBS (75 μg/ml protein). Theplates were then incubated for 30 min at 37° C. To allow thenonconjugated MAb to compete with the MAb conjugates, 50 ml of theoptimal dilution of each of the five anti-MRP MAbs conjugated to HRPOwere added. After incubation for 1 h at 37° C., plates were washed andthe bound HRPO antibody was then detected by the addition of thesubstrate 5-AS H₂ O₂ as described above. After 2 h incubation at roomtemperature, the absorbance was read. The titers of competition wereexpressed as the highest dilution showing an A₄₅₀ of 50% of the meanabsorbance of wells to which only conjugate was added. The epitopespecificity of the three anti-EF MAbs was determined with a competitionELISA similar to the one described for the anti-MRP MAbs.

SDS-PAGE and Western blotting techniques. Culture supernatants of the 22S. suis serotypes and the other microrganisms (Table 6) were separatedby SDS-PAGE on 6% polyacrylamide. For Western blot analysis, theproteins were electroblotted onto nitrocellulose by the Multiphor IINova Blot system according to the recommendations of the manufacturer(Pharmacia LKB). The blots were probed with a 1:300 dilution of mouseMAb. Bound MAbs were visualized with a 1:1000 dilution of anti-mouseimmunoglobulins conjugated with alkaline phosphatase (Zymed).

RESULTS

Direct competition ELISA. The five anti-MRP clones and the three anti-EFclones were tested for competition. Some anti-MRP clones competed witheach other. The five anti-MRP MAbs were directed against at least threedifferent epitopes: the first was recognized by MRP₁ and MRP₂, thesecond by MRP₃, and the third by MRP₄ and MRP₅. Because all threeanti-EF clones competed, they are probably directed against the sameepitope.

MRP double antibody sandwich ELISA. In an MRP DAS-ELISA using MRP₃ ascatching antibody and HRPO-MRP₁ as conjugate, each well of thepolystyrene microtiter ELISA plates was coated with 100 μl containing2.3 μg MRP₃ per well in 0.05M carbonate buffer, pH 9.6. After adsorptionfor 16 h at 37° C., coated plates were used immediately or stored at-20° C. Twofold serial dilutions of 100 μl culture supernatants, rangingfrom 1:1 to 1:128 in PBS containing 0.05% (wt/vol) Tween 80, of strainsto be tested, were added to the wells. After 1 h incubation at 37° C.,plates were washed five times with 0.05% Tween 80 in tap water, and 100μl 7.2. was added to each well. Using checker-board titrations, theoptimal dilution of catching antibody and conjugate was determined.After 1 h incubation at 37° C., the substrate 5-AS H₂ O₂ was added asdescribed above. Wells with an A₄₅₀ ≧0.2 were scored positive. To eachplate a positive control was added, consisting of 100 μl of undilutedculture supernatant of the virulent S. suis type 2 strain 4005(MRP+EF+). A negative control was also added, consisting of 100 μl ofundiluted culture supernatant of the non-virulent strain T-15 (MRP-EF-)(43).

The MRP DAS-ELISA was used to test 179 strains of S. suis type 2belonging to the three phenotypes MRP+EF+, MRP+EF-, and MRP-EF-, as waspreviously determined by SDS-PAGE and Western blot. Most strains scoredin the ELISA the same as they did in the Western blot (Table 7). AllMRP+EF+ strains were MRP-positive in the ELISA. One MRP+EF- strainscored false negative. Three of the MRP-EF- strains (6%) scored falsepositive. The sensitivity (TP/TP+FN) (TP=true positive, FN=falsenegative) of the MRP DAS-ELISA was 99% (130 out of 131 strains), thespecificity (TN/TN+FP) (TN=true negative, FP=false positive) was 94% (45out of 48 strains), and the predictive value (TP/TP+FP) was 98% (130 outof 133 strains). The MRP DAS-ELISA discriminated well between theMRP-positive and MRP-negative strains of S. suis type 2.

                  TABLE 7                                                         ______________________________________                                        Results of 179 strains of S. suis type 2 (three phenotypes)                   tested in the MRP and EF DAS-ELISAs.                                                 MRP DAS ELISA EF DAS ELISA                                                      No.       No.       No.     No.                                      phenotype                                                                              strains + strains - strains +                                                                             strains -                                ______________________________________                                        MRP+EF+  92 (100%)  0        92 (100%)                                                                              0                                       MRP+EF-  38 (97%)   1 (3%)    0      39 (100%)                                MRP-EF-   3 (6%)   45 (94%)   0      48 (100%)                                ______________________________________                                    

Titration curves of culture supernatants of strains belonging to threephenotypes of S. suis type 2, after testing in the MRP DAS-ELISA, wererecorded. The mean (± standard deviation) of the absorbances obtainedfrom the undiluted culture supernatants of the 92 MRP+EF+ isolates was1.2259 (±0.1165), the mean absorbance of the 39 MRP+EF- isolates was1.2129 (±0.2076), and the mean absorbance of the 48 MRP-EF- isolates was0.1180 (±0.2546). Therefore plates can be read visually instead ofhaving to be measured photometrically to discriminate MRP-positivestrains (phenotypes MRP+EF+ or MRP+EF-) from MRP-negative strains(phenotype MRP-EF-).

Culture supernatants of 18 of the 21 reference S. suis strains of otherserotypes had absorbances lower than 0.2. Three serotypes were positiveand had the following absorbance values: undiluted culture supernatantof serotype 3 had A₄₅₀ =0.731; culture supernatant of serotype 5 hadA₄₅₀ =0.587, and culture supernatant of serotype 15 (former Lancefieldgroup T) had A₄₅₀ =0.516. These serotypes were also positive in theWestern blot; MRP₃ apparently recognized proteins of higher molecularweight than 150 kDa in the culture supernatants of these serotypes.Absorbances of all other microorganisms listed in Table 6 were <0.2.

EF Double Antibody Sandwich ELISA. In a DAS ELISA that recognizes aspecific antigen in the test sample, two different MAbs were used, oneas catching antibody and the other as conjugate, and each recognizingdifferent epitopes on the antigen, as was done for the MRP DAS-ELISA. Inthe Western blot the EF MAbs recognize a high molecular form of EF (>150kDa) in the culture supernatants of all strains belonging to theMRP+EF-phenotype (Example 4). Therefore it is unlikely that an ELISAwith EF₂ as catching antibody can discriminate between MRP+EF+ andMRP+EF- strains. Moreover, because the three EF MABs blocked each other,we had to use EF₂ as catching antibody and the polyclonal rabbit serum(K₁₉₁) as conjugate. Some ELISAs were tested using EF₁ as catchingantibody and EF₂ or EF₃ as conjugates, and indeed these MAbs blockedeach other completely.

The procedure of the EF DAS-ELISA was essentially as that described forthe MRP DAS-ELISA. Each well of the microtiter ELISA plates was coatedwith 100 ml containing 3.3 μg of EF₂ in 0.05M carbonate buffer, pH 9.6.After adsorption, coated plates were used immediately or stored at -20°C. Twofold serial dilutions of 100 μl culture supernatants ranging from1:1 to 1:128 were used. After incubation and washings, 100 μl containing2.7 μg polyclonal Ra K₁₉₁ HRPO conjugate in PBS, pH 7.2, was added toeach well. After 1 h incubation at 37° C., the plates were developedwith substrate 5-AS H₂ O₂ as described above. Wells with an A₄₅₀ ≧0.4were scored positive. The same controls as mentioned above were used oneach plate.

The 179 S. suis type 2 strains with a predetermined protein profile weretested in the EF DAS-ELISA. Surprisingly, none of the 39 MRP+EF- strainsscored positive in this ELISA, whereas all 92 MRP+EF+ strains did (Table7). All 48 MRP-EF- strains were negative in the EF DAS-ELISA. Since noother false positive or false negative results were detected, the EFDAS-ELISA apparently discriminated reliably between the high and the lowmolecular form of EF, and hence between S. suis type 2 strains belongingto the MRP+EF+ and MRP+EF- phenotypes.

Since the direct competition ELISA had shown that the three anti-EF MAbsblocked each other, MAb EF₂ was used as catching antibody and thepolyclonal Ra K₁₉₁ serum as conjugate. Streptococcus suis type 2 strainsbelonging to the MRP+EF- phenotype, however, produce a high-molecularweight (>150 kDa) form of EF (example 4). Because MAb EF₂ does notdiscriminate between the 110-kDa EF and this high-molecular weight formin the Western blot, it was unlikely to do so in the EF DAS-ELISA.Surprisingly Mab EF₂ captured the 110-kDa EF in the culture supernatantof all MRP+EF+ strains but apparently not the higher-molecular weightform in the MRP+EF- strains (Table 7). Some MRP-EF- strains gave signalsbetween 0.2 and 0.4, which were still lower than 50% of the maximalabsorbance values and thus not high enough to be interpreted aspositive. Treating the culture supernatants with SDS before blotting mayuncover epitopes of the higher-molecular weight form of EF that are notaccessible to the EF₂ MAb in its undenaturated form. Because all MRP+EF-strains and other S. suis serotypes showed no false negative or falsepositive reactions in this ELISA, the sensitivity and specificity of thetest were considered to be 100%.

Titration curves of culture supernatants of strains belonging to threephenotypes of S. suis type 2 were recorded after testing in the EFDAS-ELISA. The mean (± standard deviation) of the absorbances obtainedfrom the undiluted culture supernatants of the 93 MRP+EF+ strains was0.8204 (±0.149), the mean absorbance of the 39 MRP+EF- strains was0.1551 (±0.046), and the mean absorbance of the 48 MRP-EF- strains was0.1061 (±0.0371). Thus, as for the MRP DAS-ELISA, plates can be readvisually to discriminate between EF-positive strains (phenotype MRP+EF+)and EF-negative strains (phenotypes MRP+EF- or MRP-EF-).

None of the 21 reference S. suis strains with a serotype other than type2 were EF-positive in the ELISA. Some other bacterial species hadpositive absorbance values: Streptococcus Lancefield group G (A₄₅₀=0.445), group L (A₄₅₀ =0.348), Streptococcus equi (A₄₅₀ =0.671), andStaphylococcus aureus (A₄₅₀ =0.718).

EXAMPLE 7

Differentiation between pathogenic and non-pathogenic strains ofStreptococcus suis type 2 by using polymerase chain reaction (PCR).

MATERIALS AND METHODS.

Bacteria and growth conditions. Thirteen strains of S. suis type 2 wereselected to examine whether the Polymerase Chain Reaction (PCR) method(36) could be useful to differentiate between the three phenotypes of S.suis type 2. Pathogenicity and the expression of the MRP and EF proteinsof these strains were determined in Examples 4 and 5. Strains were grownovernight at 37° C. on Columbia blood agar base (code CM 331, Oxoid)containing 6% horse blood. S. suis type 2 colonies were inoculated in 10ml Todd-Hewitt broth (code CM 189, Oxoid), and grown overnight at 37° C.

DNA Isolations. DNA of overnight grown cultures was isolated asdescribed by Maniatis et. al (28). DNA was diluted to 10 ng/μl indistilled water before use in the PCR.

Clinical specimens. Nose swabs and tonsillar tissues were obtained postmortem from sows at slaughter. Nose swabs were inoculated on bloodplates. S. suis type 2 strains were isolated from tonsils as describedbefore (27).

Sample preparation. Clinical specimens for the PCR were prepared by themethod described by Boom et. al (4), with some minor modifications: Thespecimens were added to 900 μl L6 lysis buffer plus 40 μl diatom earthsolution in an Eppendorf tube [L6 buffer is 100 ml 0.1M TRIS HCl (pH6.4) plus 120 g guanidine (iso)thiocyanate (GuSCN, Fluka cat nr. 50990)plus 22 ml 0.2M EDTA (pH 8.0) plus 2.6 g Triton X-100. Diatom earthsolution is 10 g Diatom earth (Janssen Chimica Cat. nr. 17.346.80) in 50ml distilled water plus 500 μl 32% (w/v) HCl]. The clinical specimenswere incubated overnight in L6 buffer in the dark at room temperature.150 μl of the solution was pipetted in wells of microtiter platescontaining Durapore membranes (Multiscreen MAHV N45, Millipore). Themicrotiter plate was put on the vacuum manifold (MAVM 09600, Millipore),and the samples were washed 5 times with 200 μl L2 washing solution (L2buffer is 100 ml 0.1M Tris-HCl (pH 6.4) plus 120 g GuSCN), 5 times with200 μl 70% ethanol, and once with 200 μl aceton. The filters were notallowed to run dry between the wash steps. The bottom of the microtiterplate was dried on a tissue and the samples were dried completely for 15minutes at 56° C. 75 μl PCR buffer (see below) was added to theindividual wells. The plate was incubated for 15 minutes at 56° C. Themicrotiter plate was again put on the vacuum manifold, with a standardmicrotiter plate (Micronic) beneath the Durapore plate. Vacuum wasapplied, and the PCR buffer, containing the DNA was collected in thelower microtiter plate, whereas the diatom earth remained on theDurapore filters.

PCR assay. The PCR contained 10 ng purified DNA or 25 μl clinicalspecimen in a total volume of 50 μl. The reaction mixtures contained 10mM Tris-HCl (pH 9.0), 2 mM MgCl₂, 50 mM KCl, 0.01% gelatin, 0.2 mM ofeach of the four deoxynucleotide triphosphates, 1 μM of each of the fourprimers and 0.5 U of Amplitaq polymerase (Perkin Elmer Cetus, Norwalk,Conn.), and was overlaid with 2 drops of paraffine oil. DNAamplification was carried out in a Perkin Elmer Thermal Cycler for 25 or40 cycles: 1 minute 94° C., 1 minute 55° C., and 2 minutes 72° C. Ten to20 μl of the amplified DNA was analysed on a 1.5% agarose gel, thatcontained ethidium bromide.

PCR primers. The sequences of the oligonucleotides used in the PCR were:p15. SEQ ID NO 3 base pairs 1403-1425: 5'-GGT ATA CCT TGC TGG TAC GGT TC-3', p16: SEQ ID NO: 3 base pairs 1914-1934: 5'-AGT CTC TAC AGC TGT AGCTGG -3'(which correspond to the complement), p-34: SEQ ID NO: 2 basepairs 2890-2908: 5'-GTT GAA AAC AAA GCA TTC G -3', and p-35: SEQ ID NO 2base pairs 3229-3249: 5'- CTT CGA CAA AAT GTC AGA TTC -3'. Theoligonucleotides p-15 and p-16 correspond to the indicated positions inthe S. suis type 2 mrp gene (Example 3, SEQ ID NO:3). Theoligonucleotides p-34 and p-35 correspond to the indicated positions andin the S. suis type 2 ef* gene (Example 2, SEQ ID NO:2). Primers weresynthesized on an Applied Biosystem synthesizer type 381A following themanufacturers protocol.

RESULTS

Specificity of PCR. Within the mrp and ef* genes (cf. Examples 3 and 2),two regions (designated as m-VI and e-V) were determined that could beused to differentiate between the three phenotypes of S. suis type 2strains (see also Example 8). Primers based on the m-VI region (p-15 andp-16), and the e-V region (p-34 and p-35) were used in a PCR. Theprimers p-15 and p-16 amplified a 532 bp fragment in the m-VI region.The primers p-34 and p-35 amplified a 360 bp fragment in the e-V region.Chromosomal DNA of 4 MRP⁺ EF⁺, 4 MRP⁺ EF* and 5 MRP⁻ EF⁻ strains wasused in a PCR with these primers (see FIG. 15). After 25 cycli theamplified fragments were analysed on an agarose gel. A 532 bp fragmentwas amplified from DNA of MRP⁺ EF⁺ strains. A 532 bp fragment as well asa 360 bp fragment were amplified from DNA MRP⁺ EF* strains. In contrast,neither the 532 bp nor the 360 bp fragment was amplified from DNA ofMRP^(-EF) ⁻ strains. These data show that this PCR can be used todifferentiate between the three phenotypes of S. suis type 2.

The phenotypes of 82 strains of S. suis type 2, isolated from thetonsils of 37 healthy sows at slaughter, were determined by Westernblotting (Example 4), ELISA (Example 6), hybridization experiments withDNA probes m-VI and e-V (Example 8), and by PCR. 79 strains, isolatedfrom 36 of the 37 sows were classified identical by the four methods(96.3%). 3 strains, isolated from one sow, were classified as MRP⁺ EF*by the PCR and DNA hybridization experiments and as MRP⁻ EF⁻ by Westernblotting and ELISA. These results indicate that the PCR is a usefulalternative to determine the phenotype of a S. suis type 2 strain.

Sensitivity of PCR. Purified chromosomal DNA of a MRP⁺ EF* S. suis type2 strain was diluted in distilled water and used directly in the PCR.After 40 cycli of PCR, 25 fg DNA was detected. This indicates that DNAof 14 cells, after amplification by PCR, could be detected on an agarosegel, based on data that a Streptococcal cel contains about 1.75 fg DNA(35). The sensitivity of the PCR on whole cells was determined.Therefore, MRP⁺ EF* cells were diluted in phosfate buffered saline (PBS(pH 7.2); 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂ HPO₄, 2.8 mM KH₂ PO₄) andprepared for PCR as described above. Amplified fragments could still bedetected in samples that contained about 50 cells prior to the PCR (40cycli).

The PCR can be used directly on clinical material. Serial dilutions ofS. suis type 2 cells were added to nose swabs. It was found thatamplified fragments can still be detected in samples that contain about50 cells prior to the PCR.

EXAMPLE 8

Differentiation between pathogenic and non-pathogenic strains of S. suistype 2 using DNA probes.

MATERIALS AND METHODS

Bacteria. Thirteen strains of S. suis type 2 (4 MRP⁺ EF* strains and 5MRP⁻ EF⁻ strains) were selected to examine whether regions of the mrp,ef, and ef* genes could be useful to differentiate between the threephenotypes of S. suis type 2. Except for strain 16, pathogenicity ofthese strains was tested in an infection experiment of piglets (Example5).

170 strains of S. suis type 2 were obtained from three sources: Fromorgans of diseased pigs (103 strains), from tonsils of healthy pigs atslaughter (40 strains) and from human patients (27 strains). Referencestrains of S. suis serotypes 1 to 22 (15), 21 other Streptococci speciesand 45 other bacterial strains (38 different species, DLO CentralVeterinary Institute, Table 8) were used to test the specificity of themrp and ef probes.

Media. E. coli JM101 strains were grown in LB broth (30). Ampicillin wasadded as needed to a final concentration of 50 μg/ml. All otherbacterial strains were grown overnight at 37° C. on Columbia blood agarbase (code CM 331, Oxoid) containing 6% horse blood. Overnight growncolonies were incubated in 10 ml Todd-Hewitt broth (code CM 189, Oxoid),and grown overnight at 37° C.

DNA isolations and manipulations. Chromosomal DNA isolations and routineDNA techniques were performed as described by Maniatis et al (28). Crudelysates were made as follows: overnight grown cultures were centrifugedat 4000× g for 10 minutes, and the pellet fraction was resuspended in500 to 1000 μl TEG-lysozym buffer (25 mM TRIS.Cl pH 8.0, 10 mM EDTA, 50mM glucose and 1 mg/ml lysozym). After 30 minutes at 25° C., the sampleswere used in the dot-blot assay.

Probes. The plasmids pMR11, pEF2-19 and pEF17-7 (cf. Examples 1, 2, 3)were used to generate subclones into pKUN19 (24). Fragments ofappropriate subclones were isolated from preparative agarose gels withthe gene-clean kit (Bio 101 Inc., La Jolla, USA). Purified fragmentswere subsequently labeled with α-³² P dCTP (3000 Ci/mMol, Amersham) withthe random primed labeling kit (Boehringer GmbH) following themanufacturers protocol and used as probes.

Southern hybridizations. Chromosomal DNA of the 13 selected S. suis 2strains (1 μg DNA) was spotted on Gene-screen nylon membrane(New-England Nuclear Corp., Boston, USA). The membranes were incubatedwith the ³² P-labeled mrp and ef probes as recommended by themanufacturer. After overnight hybridization, the filters were washedtwice with 2× SSC for 5 minutes at room temperature, and twice with 0.1×SSC plus 0.5% (SDS) for 30 minutes at 65° C. (1× SSC=0.15M NaCl plus0.015M Sodium Citrate). For the group of 170 S. suis 2 strains, the 22reference strains of S. suis type 1 to 22, and the group of otherStreptococci and other bacteria, 20 μl of a DNA or crude lysate samplewas dotted on Zeta probe nylon membrane (Biorad) with a dot blotapparatus (Bethesda Research Laboratories).

The membranes were incubated with the ³² P-labeled mrp and ef probes asrecommended by the manufacturer. After overnight hybridization, themembranes were washed twice in 40 mM Na phosphate buffer, pH 7.2 plus 5%SDS plus 1 mM EDTA for 30 minutes at 65° C. and twice in 40 mM Naphosphate buffer, pH 7.2 plus 1% SDS plus 1 mM EDTA for 30 minutes at65° C. All (pre)hybridizations were carried out in a hybridization oven(Hybaid).

RESULTS

Mrp probes. Chromosomal DNA of the 3 phenotypes of S. suis type 2 washybridized to different regions of the mrp gene. Six different mrpprobes were used (schematically shown in FIG. 14a). The EcoRI-SnaBIfragment, m-I, contained the entire mrp encoding region. The m-II,m-III, m-IV and m-V probes contained different regions of the mrp gene(see FIG. 16). The MRP⁺ EF⁺ and the MRP⁺ EF* strains strongly hybridizedwith all mrp probes. In addition, the m-I, m-II, m-IV and m-V probesstrongly hybridized with 4 of the 5 MRP⁻ EF⁻ strains. One MRP⁻ EF⁻strain (strain 25) did not hybridize with any of the mrp probes. Thesedata indicate that 4 MRP⁻ EF⁻ strains contained large regions homologousto the mrp gene of strain D282, whereas strain 25 lacked the entire mrpgene. These 4 MRP⁻ EF⁻ strains, however, hybridized only weakly withprobe m-III, indicating that only a small part of probe m-III washomologous to their DNA. A probe m-VI was constructed by removing 385 bpat the 5', and 325 bp at the 3' ends of probe m-III. The 5 MRP⁻ EF⁻strains did not hybridize at all with probe m-VI, indicating that thesestrains lacked the region homologous to the m-VI probe. Therefore, probem-VI can be used to differentiate between MRP⁺ and MRP⁻ strains.

Ef and ef* probes. Chromosomal DNA of the 3 phenotypes S. suis type 2was hybridized to different regions of the ef gene. Four different efprobes (schematically shown in FIG. 14b) were used. All MRP⁺ EF⁺ andMRP⁺ EF* strains and 1 MRP⁻ EF⁻ strain hybridized with all ef probes. Incontrast, 4 MRP⁻ EF⁻ strains did not hybridize with any of the efprobes. These data indicate that most of these MRP⁻ EF⁻ strains lackedthe entire region homologous to the ef gene, whereas 1 MRP⁻ EF⁻ strainseemed to contain the entire region homologous to the ef gene.Therefore, the probes e-I to e-IV could not been used to differentiatebetween the 3 phenotypes.

Since the gene encoding the EF* proteins contain a DNA fragment which isabsent in the gene encoding the EF protein, part of this extra DNA wasselected as a probe (FIG. 14c, probe e-V). Probe e-V hybridized with allMRP⁺ EF* strains. On the contrary, none of the MRP⁺ EF⁺ and MRP⁻ EF⁻strains hybridized with the e-V probe. These data suggest that the MRP⁺EF⁺ and MRP⁻ EF⁻ strains lacked the region homologous to e-V. Probe e-Vis thus specific for MRP⁺ EF* strains.

Therefore, if m-VI and e-V are used in complementary hybridizationstudies, a differentiation between the three phenotypes of S. suis type2 will be possible. If S. suis type 2 strains hybridize with probe m-VIand e-V, these strains belong to the MRP⁺ EF* phenotype. If S. suis type2 strains hybridize with m-VI but not with e-V, these strains belong tothe MRP⁺ EF⁺ phenotype, and finally if strains do not hybridize withm-VI and e-V, these strains belong to the MRP⁻ EF⁻ phenotype.

The mrp, ef and ef* probes were tested on 170 other strains of S. suistype 2. 88 strains had a MRP⁺ EF⁺ phenotype, 37 strains a MRP⁺ EF*phenotype and 45 strains had a MRP⁻ EF⁻ phenotype. In accord with thedata presented above, all MRP⁺ EF⁺ strains hybridized with the probesm-I to m-VI and e-I to e-IV, but none hybridized with probe e-V.Moreover, all the 37 MRP⁺ EF* strains hybridized with all the probes.Only two of the 45 MRP⁻ EF⁻ strains, however, hybridized with probe m-VIand e-V and would therefore wrongly be classified as MRP⁺ EF* strains.Therefore, by using m-VI and e-V, the phenotype of a S. suis type 2strain can be predicted with a very high probability (168/170; 98.8%).

Specificity of the m-VI and e-V probes. DNA of the reference strains ofS. suis serotype 1 to serotype 22 was tested for hybridization withprobes m-VI and e-V. It was found that S. suis type 2 (strain 735), 4, 5and 14 hybridized with the m-VI probe and that type 1/2, 2, 4, 5, 6, 14and 15 hybridized with the e-V probe. These data suggest that the mrpand ef genes are not specific for S. suis type 2, but that homologoussequences are present in several serotypes. Based on these data,serotypes 2, 4, 5 and 14 would be classified as MRP⁺ EF* strains,whereas serotypes 1/2, 6 and 15 would be classified as MRP⁻ EF⁻ strains.

Chromosomal DNA from swine pathogens and several common bacteria wastested with the probes m-I, m-VI, e-III and e-V. The species tested arelisted in Table 8. Although some species hybridized with probe m-I(Escherichia coli, Klebsiella oxytoca, K. pneumoniae and Salmonellatyphimurium), none hybridized with the probes m-VI, e-III and e-V. Thesedata shown that although in some species parts of the mrp genes arefound, the probes m-VI and e-V are specific for S. suis. Hence, theprobes m-VI and e-V have potential diagnostic value.

                  TABLE 8                                                         ______________________________________                                        List of other species on which the probes were tested for                     specificity.                                                                  ______________________________________                                        Streptococcus species                                                         S. agalactiae     S. equi                                                     S. equisimilis porcine                                                                          S. zooepidemicus                                            S. dysgalactiae   Enterococcus faecalis                                       E. liquefaciens   E. zymogenes                                                E. faecium        S. group E                                                  S. milleri III    S. bovis                                                    S. pyogenes humanis                                                                             S. uburis                                                   S. animale G      S. group G                                                  S. group L biotype I                                                                            S. group L biotype II                                       S. group P        S. group Q                                                  S. sanguis                                                                    Other Bacteria                                                                Actinobacillus pleuropneumoniae                                                                 Actinobacillus viridans                                     Actinobacillus suis                                                                             Aeromonas hydrophila                                        Actinomyces pyogenes                                                                            Bacillus licheniformis                                      Bacillus cereus   Bordetella bronchiseptica                                   Bacillus subtilis Brucella suis biotype II                                    Brucella suis biotype I                                                                         Campylobacter faecalis                                      Campylobacter coli                                                                              Candida albicans                                            Campylobacter jejuni                                                                            Erysipelothrix rhusiopathiae                                Clostridium perfringens                                                                         Klebsiella oxytoca                                          A non-toxic                                                                   Clostridium perfringens                                                                         Listeria monocytogenes                                      A toxic                                                                       Escherichia coli  Micrococcus luteus                                          Haemophilus parasuis                                                                            Mycoplasma hyopneumoniae                                    Klebsiella pneumoniae                                                                           Mycoplasma hyosynoviae                                      Micrococcus strain 3551                                                                         Pasteurella multocida                                       Mycobacterium avium serovar2                                                                    Salmonella typhimurium                                      Mycoplasma hyorhinis                                                                            Staphylococcus aureus                                       Pseudomonas aeruginosa                                                                          Staphylococcus hyicus                                       Pasteurella vulgaris                                                                            hyicus                                                      Serratia liquefaciens                                                         Staphylococcus epidermidis                                                    Yersinia enterocolitica                                                       ______________________________________                                    

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4376 base pairs                                                   (B) TYPE: Nucleic acid with corresponding amino acids                         (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus suis type II (pathogenic)                         (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Extracellular protein factor (EF) gene                 (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 66 to 71                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 89 to 94                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 153 to 158                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 176 to 181                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: ribosome binding site                                           (B) LOCATION: bp 350 to 356                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: signal peptide                                                  (B) LOCATION: bp 361 to 498                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: mature peptide                                                  (B) LOCATION: bp 499 to 2890                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: dyad symmetry regions                                           (B) LOCATION: from bp 4186 to 4198 and from bp 4203 to 4215                   (ix) FEATURE:                                                                 (A) NAME/KEY: dyad symmetry regions                                           (B) LOCATION: from bp 4243 to 4257 and from bp 4263 to 4276                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTGAACAACTTAAAACTAGTTAGTTTTGTTTAAAATGTAATTGAATTGTCTTTTTAAGTA60                GGCTGTTTACACGATATTTGTCTTCCTTTATATAAATATGATAGATTTTCAGTAAATTTT120               TCAAAAAAACCTCAAAAATAACAGATTTTTTCTTGTATCTTTGAGGCATAAGGAGTATAA180               TGGTGACGGTATTCAAGTAGAAATTTTATATACTCTTGATGAAAACATTCTGTCTACTTT240               AAAATAAATAATCTACTGGGTATCCTTCTGCTAAGTTTTTAAAGCAGGAGGTGTGTTTTT300               GTACATGGTGTTACAGGAACCAGAAATGATCGATTCGCCAGTAAAATATAGGAGGATATC360               ATGTCTTATAAAGATATGTTCAGAAAAGAACAACGTTTTTCTTTTCGT408                           MetSerTyrLysAspMetPheArgLysGluGlnArgPheSerPheArg                              45-40-35                                                                      AAATTTAGCTTTGGTCTAGCTTCGGCAGTCATTGCAAACGTTATTTTG456                           LysPheSerPheGlyLeuAlaSerAlaValIleAlaAsnValIleLeu                              30-25-20-15                                                                   GGAGGAGCAATCGCAAACAGCCCTGTTGTTCATGCTAACACAGTGACA504                           GlyGlyAlaIleAlaAsnSerProValValHisAlaAsnThrValThr                              10-51                                                                         GAAGCAGAGACAGCTGTAGCACCAGCTAACCAAGACCTTGGAAATGAG552                           GluAlaGluThrAlaValAlaProAlaAsnGlnAspLeuGlyAsnGlu                              51015                                                                         ACTAAAACGGAAGAAGAACCCAAGGAACCAATCGAAGCAGTTCGCACG600                           ThrLysThrGluGluGluProLysGluProIleGluAlaValArgThr                              202530                                                                        GACATGGAAAACCGTGCAGCTGAAATCTTGCCGGAGGCGCTGAATGCT648                           AspMetGluAsnArgAlaAlaGluIleLeuProGluAlaLeuAsnAla                              35404550                                                                      AGTGTAACAAACCAAGCACCAGTTATTCCGACTATTGGAGATCTTCCT696                           SerValThrAsnGlnAlaProValIleProThrIleGlyAspLeuPro                              556065                                                                        AAAGATGCGAGTGGTCAGAATGTTCATGGTAAGGCAACGGATAATAAG744                           LysAspAlaSerGlyGlnAsnValHisGlyLysAlaThrAspAsnLys                              707580                                                                        ATTTATCGTGTTGTATACGTTTTTGGTAATGTAGCAGGGACTACGGAG792                           IleTyrArgValValTyrValPheGlyAsnValAlaGlyThrThrGlu                              859095                                                                        ACAGAAGATGGTAAACAAAATGTTGCTCCAACATTTAACAGAAATGAT840                           ThrGluAspGlyLysGlnAsnValAlaProThrPheAsnArgAsnAsp                              100105110                                                                     GCAACTAAAACTTTTCCAATCACAGATCCAGATAGCGACATTCAAACT888                           AlaThrLysThrPheProIleThrAspProAspSerAspIleGlnThr                              115120125130                                                                  ATTTCATACGAAGTTCCAGCTGATATTGCAAGCTATACCTTGGATGAT936                           IleSerTyrGluValProAlaAspIleAlaSerTyrThrLeuAspAsp                              135140145                                                                     CCAAACTCAATTGTTACTAATGGCACCTCACCTGGTCCAGTATCTTAC984                           ProAsnSerIleValThrAsnGlyThrSerProGlyProValSerTyr                              150155160                                                                     TTAGATGGTCCAAATGGGTCAGCCACTCTCACACAAGATGGTTATCTA1032                          LeuAspGlyProAsnGlySerAlaThrLeuThrGlnAspGlyTyrLeu                              165170175                                                                     ACAGGAAGTTTCCCTTGGGGAGCAGGAGACCTAGCTGGTCGTCGGATT1080                          ThrGlySerPheProTrpGlyAlaGlyAspLeuAlaGlyArgArgIle                              180185190                                                                     AAAGTGACGGATGCCACTGGTAATACTACTAAGAGTAATCCGTTCTAT1128                          LysValThrAspAlaThrGlyAsnThrThrLysSerAsnProPheTyr                              195200205210                                                                  ATGGTTGCATATACAGTCAAGCCAGTAGATGATAAACCTCTAGCAGTA1176                          MetValAlaTyrThrValLysProValAspAspLysProLeuAlaVal                              215220225                                                                     TCAAACTCTTCTGAGCTGACGGAACAGGCTATTTTTGATAAGTTGGTT1224                          SerAsnSerSerGluLeuThrGluGlnAlaIlePheAspLysLeuVal                              230235240                                                                     GTCGATAAGTCTGCTAAAACAACTTCAAATAGCGCTCTTGTAATTGAT1272                          ValAspLysSerAlaLysThrThrSerAsnSerAlaLeuValIleAsp                              245250255                                                                     TCTAGCAACTACAAACATTCAATTGCAGGTTATCGTACCGTAAATTCT1320                          SerSerAsnTyrLysHisSerIleAlaGlyTyrArgThrValAsnSer                              260265270                                                                     GATGGCACAAAAACAGAAACAGTAGAGGAAACAAATCTATCTGATTTC1368                          AspGlyThrLysThrGluThrValGluGluThrAsnLeuSerAspPhe                              275280285290                                                                  CCAACTGAAGGTAAATACGAAGTTCGAGTAAAAACAACCAATGTTTAC1416                          ProThrGluGlyLysTyrGluValArgValLysThrThrAsnValTyr                              295300305                                                                     GGTCAAACTATCTACAACTGGATTCCTGTAAATGCCTATAAGTTGGAC1464                          GlyGlnThrIleTyrAsnTrpIleProValAsnAlaTyrLysLeuAsp                              310315320                                                                     ACAGCGAAGGATGCTGAAATTCGGAAGTATACAGACAACCAAGCCCCA1512                          ThrAlaLysAspAlaGluIleArgLysTyrThrAspAsnGlnAlaPro                              325330335                                                                     ATTCATGCTATAATGCAAATTGGTCAAGCTGGAGAAAAGGCAGCAGTT1560                          IleHisAlaIleMetGlnIleGlyGlnAlaGlyGluLysAlaAlaVal                              340345350                                                                     ATATTGAAGGATATTCCATCCGATTTCAGTATTGAAAACTTCAATTTG1608                          IleLeuLysAspIleProSerAspPheSerIleGluAsnPheAsnLeu                              355360365370                                                                  AAAGATGGTGTAGCAGATGAGCTTGCTAAACGTAACTTGGAATTTGTA1656                          LysAspGlyValAlaAspGluLeuAlaLysArgAsnLeuGluPheVal                              375380385                                                                     AGAAATGATGCAGTGGCGACAACTGATACTGATGGAGATGGCGCCAAA1704                          ArgAsnAspAlaValAlaThrThrAspThrAspGlyAspGlyAlaLys                              390395400                                                                     GAAGGAATTGTTGGATATATTCAACCAAAAACTGGCGGTGCAAACAGT1752                          GluGlyIleValGlyTyrIleGlnProLysThrGlyGlyAlaAsnSer                              405410415                                                                     GGGGTAGCCACTTATACAGGATCAAATAATCTTACTTATGGCTTCACT1800                          GlyValAlaThrTyrThrGlySerAsnAsnLeuThrTyrGlyPheThr                              420425430                                                                     TACAAAGCTGTTGAGACAAAAGATAAGGCGAATGCCACAGAGGCTAAA1848                          TyrLysAlaValGluThrLysAspLysAlaAsnAlaThrGluAlaLys                              435440445450                                                                  ACTCTCGAATTAGATTACACCATCTTATTCATAGATACTAAAGCACCA1896                          ThrLeuGluLeuAspTyrThrIleLeuPheIleAspThrLysAlaPro                              455460465                                                                     GTCATGACACCTAAATCAGAGTACATCCGTTTTGTTGGTGAAGAGTAT1944                          ValMetThrProLysSerGluTyrIleArgPheValGlyGluGluTyr                              470475480                                                                     ACGGTTAGCGTCCCAGGTACGGATAACGCCTTCCTTAATACCGGCAAA1992                          ThrValSerValProGlyThrAspAsnAlaPheLeuAsnThrGlyLys                              485490495                                                                     CTAAATGGAACTCTCTCAATTTTGAAAGATGGAGAGTCAGGTTCTCTT2040                          LeuAsnGlyThrLeuSerIleLeuLysAspGlyGluSerGlySerLeu                              500505510                                                                     GTATCATCAGACTTAGGTACAAACACTAAGATTACTTCAGAACTGGAT2088                          ValSerSerAspLeuGlyThrAsnThrLysIleThrSerGluLeuAsp                              515520525530                                                                  CCTACGGGAGCAACTGCAAACCAAGGAGATGACGGTCAATCTTCAACT2136                          ProThrGlyAlaThrAlaAsnGlnGlyAspAspGlyGlnSerSerThr                              535540545                                                                     AAGTTTAACGTTAAGATTACAGGTACCGGACCTGCTACAGAAGGTACC2184                          LysPheAsnValLysIleThrGlyThrGlyProAlaThrGluGlyThr                              550555560                                                                     GGCACTTATAAGCTTCGTGTTGGAGAAGATAACTATCCTTTTGGTCCA2232                          GlyThrTyrLysLeuArgValGlyGluAspAsnTyrProPheGlyPro                              565570575                                                                     GAGGGGAAACTTGTTGATGGAAATAAACCAGAAAATGTAGGTTTGACA2280                          GluGlyLysLeuValAspGlyAsnLysProGluAsnValGlyLeuThr                              580585590                                                                     TCTGTAAAAGTTACCTTCGTAAAACATGCTACGGTGTCAACACCAGTT2328                          SerValLysValThrPheValLysHisAlaThrValSerThrProVal                              595600605610                                                                  TCTGTTGAAAATCCAGCTAACTTAACGCCAGAAGAAAAAGCCGCAGTT2376                          SerValGluAsnProAlaAsnLeuThrProGluGluLysAlaAlaVal                              615620625                                                                     ATTGCTCAAATCAAGAAAGACAACGCAGACAACGAAAGATTGAAGGGC2424                          IleAlaGlnIleLysLysAspAsnAlaAspAsnGluArgLeuLysGly                              630635640                                                                     TTGCCAGATTCAGCATTTACAGTTAACTCAGATGGTACTGTGTCAGTT2472                          LeuProAspSerAlaPheThrValAsnSerAspGlyThrValSerVal                              645650655                                                                     GACTACAGTGCCGGTGGTGTCAATGTTGATGGTGCGACAGACATTATT2520                          AspTyrSerAlaGlyGlyValAsnValAspGlyAlaThrAspIleIle                              660665670                                                                     AAGAATGCTACCACAAACTTGGCAGATACACGGAATGAAGCAAAAGCA2568                          LysAsnAlaThrThrAsnLeuAlaAspThrArgAsnGluAlaLysAla                              675680685690                                                                  GAAATCGACACAAAATTAGCTGAACATAAAAAAGCTATCGAAGCAAAA2616                          GluIleAspThrLysLeuAlaGluHisLysLysAlaIleGluAlaLys                              695700705                                                                     CGGGATGAAGCGTTTTCTAAAATTGATGATGACATTTCCTTGAGAGCA2664                          ArgAspGluAlaPheSerLysIleAspAspAspIleSerLeuArgAla                              710715720                                                                     GAACAGAGACAGGCTGCTAAGGATGCCGTTGCTGCAGCTGCTGGGGAT2712                          GluGlnArgGlnAlaAlaLysAspAlaValAlaAlaAlaAlaGlyAsp                              725730735                                                                     GCTTTGAAAGAATTAGACAACAAGGCGACAGAAGCAAAAGAAAAAATT2760                          AlaLeuLysGluLeuAspAsnLysAlaThrGluAlaLysGluLysIle                              740745750                                                                     GATAAAGCTACGACGGCCTCAGAAATCAATGATGCTAAGACTAATGGT2808                          AspLysAlaThrThrAlaSerGluIleAsnAspAlaLysThrAsnGly                              755760765770                                                                  GAGATTAATCTGGACAGTGCAGAAGCAGTAGGCGAAAAAGCTATTAAC2856                          GluIleAsnLeuAspSerAlaGluAlaValGlyGluLysAlaIleAsn                              775780785                                                                     CAGTCGAAGCGCAATCGGCAGAGGACAAAGGCGTAGGTTCAATCGCC2903                           GlnSerLysArgAsnArgGlnArgThrLysAla                                             790795                                                                        CAAGATGTTCTTGACGCAGCGAAACAAGATGCTAAGAATAAGATTGCT2951                          GlnAspValLeuAspAlaAlaLysGlnAspAlaLysAsnLysIleAla                              800805810                                                                     AAAGAATCCGACGCTGCTAAGTCAGCCATTGACGCGAATCCAAACTTG2999                          LysGluSerAspAlaAlaLysSerAlaIleAspAlaAsnProAsnLeu                              815820825                                                                     ACAGATGCAGAGAAGGAATCAGCTAAGAAAGCGGTAGATGCAGATGCT3047                          ThrAspAlaGluLysGluSerAlaLysLysAlaValAspAlaAspAla                              830835840845                                                                  AAAGCTGCGACAGATGCAATTGATGCTTCAACAAGTCCAGTCGAAGCG3095                          LysAlaAlaThrAspAlaIleAspAlaSerThrSerProValGluAla                              850855860                                                                     CAATCGGCAGAGGACAAAGGCGTAGGCGCCATCGCCAAAGACATTCTT3143                          GlnSerAlaGluAspLysGlyValGlyAlaIleAlaLysAspIleLeu                              865870875                                                                     GATGCCGCGAAACAAGATGCTAAGAACAAGATTGCTAAAGAGGCAGAA3191                          AspAlaAlaLysGlnAspAlaLysAsnLysIleAlaLysGluAlaGlu                              880885890                                                                     TCCGCTAAGTCAGTCATTGACTCCAATCCGAACTTGACAGATGCAGCT3239                          SerAlaLysSerValIleAspSerAsnProAsnLeuThrAspAlaAla                              895900905                                                                     AAGGAAGCGGCTAAATCTGAAATTGATAAAGCTGTTGAGGAAGCGATT3287                          LysGluAlaAlaLysSerGluIleAspLysAlaValGluGluAlaIle                              910915920925                                                                  GTTTTAATCAATGGTGTTAGAACTTATCAAGAGTTGGAAAAAATCAAA3335                          ValLeuIleAsnGlyValArgThrTyrGlnGluLeuGluLysIleLys                              930935940                                                                     CTTCCAATGGCAGCTCTAATTAAACCAGCTGCGAAAGTAACACCAGTG3383                          LeuProMetAlaAlaLeuIleLysProAlaAlaLysValThrProVal                              945950955                                                                     GTTGATCCAAATAACTTGACTGAAAAAGAAATTGCTCGTATCAAGGCA3431                          ValAspProAsnAsnLeuThrGluLysGluIleAlaArgIleLysAla                              960965970                                                                     TTCCTTAAAGAGAACAATAACCTCCCATAAGGAACAGAGATTAATGTT3479                          PheLeuLysGluAsnAsnAsnLeuProGlyThrGluIleAsnVal                                 975980985                                                                     TCTAAAGATGCTTCAGTGACAATTAAATATCCAGATGGAACTATTGAT3527                          SerLysAspAlaSerValThrIleLysTyrProAspGlyThrIleAsp                              9909951000                                                                    TTGCTATCACCAGTAGAAGTTGTGAAGCAGGCAGATAAAACTGCTCCT3575                          LeuLeuSerProValGluValValLysGlnAlaAspLysThrAlaPro                              1005101010151020                                                              ACGGTCGCAAATGATGGCAAAGGTAATATTGTGATTGTACCGTCTGAA3623                          ThrValAlaAsnAspGlyLysGlyAsnIleValIleValProSerGlu                              102510301035                                                                  AAAGCTGTTGAGCTTGTTGTTTCATACGTAGATAACAATGGTAAGTCG3671                          LysAlaValGluLeuValValSerTyrValAspAsnAsnGlyLysSer                              104010451050                                                                  CAAACTGTAGTTGTTACGAAAGGTACGGATGGTTTATGGACAGCAAGT3719                          GlnThrValValValThrLysGlyThrAspGlyLeuTrpThrAlaSer                              105510601065                                                                  AATACAGTGGTGATTGTGGACCCTGTGACTGGGCAAGTAATCGTTCCA3767                          AsnThrValValIleValAspProValThrGlyGlnValIleValPro                              107010751080                                                                  GGTTCTGTTATTAAGCCAGGTACAGTTGTTACAGCATACTCTAAAGAC3815                          GlySerValIleLysProGlyThrValValThrAlaTyrSerLysAsp                              1085109010951100                                                              GAGGTTGGAAATAGTTCTGATTCAGCAGAAGCTGAAGTTGTAGCAGTA3863                          GluValGlyAsnSerSerAspSerAlaGluAlaGluValValAlaVal                              110511101115                                                                  GACGAAAATAATTCTGCAGCAGGAGTGAAAGTTAAATCAGTTACTACA3911                          AspGluAsnAsnSerAlaAlaGlyValLysValLysSerValThrThr                              112011251130                                                                  AATGCTAATAATGTTGAGAAGAAAGCTAAGCAATTACCGAATACTGGT3959                          AsnAlaAsnAsnValGluLysLysAlaLysGlnLeuProAsnThrGly                              113511401145                                                                  GAGGAAGCAAATTCAGCAACTTCACTCGGATTAGTAGCTCTTGGACTC4007                          GluGluAlaAsnSerAlaThrSerLeuGlyLeuValAlaLeuGlyLeu                              115011551160                                                                  GGATTAGCACTTCTTGCAGCAAAGAGAAGAAGAGACGAAGAAGCTTAA4055                          GlyLeuAlaLeuLeuAlaAlaLysArgArgArgAspGluGluAla                                 116511701175                                                                  GATAAGCTCTTCCTCAGAACTCTTTTGGAAGCCGCAATTTTCCTAGAAGATAGTAGTATG4115              ATACTCTTTCATAGCAAGGAAATTCCCTCGCTATGATTGGTAGGTATCAGTTATTATCTA4175              TCGAACCCCCAAAATCCAAAGTCATTCGACTTTGGATTTTTTTGATACGACATGCTCGTC4235              ATACCTAAAAAACAGCCTTCTCTTGCCGAGAGGCTGTTTTTCATGCTTTTAATCTAAAAG4295              TCTGCGGACGTTTTTTCAATAAAATCCAGTAACCGATGCTAACATAGGCAATCATAGCTA4355              GGGAAACCAGCAGGATATAGG4376                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6744 base pairs                                                   (B) TYPE: Nucleic acid with corresponding amino acids                         (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus suis type II (pathogenic)                         (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Extracellular factor related protein                   (EF*) gene                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 66 to 71                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 89 to 94                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 153 to 158                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 176 to 181                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: ribosome binding site                                           (B) LOCATION: bp 350 to 356                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: signal peptide                                                  (B) LOCATION: bp 361 to 498                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: start of repetitive units R1-R11                                (B) LOCATION: bp 2869, 3097, 3292, 3520, 4087, 4381, 4609,                    4837, 5065, 5293, 5521:                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: start of repetitive Asn--Pro--Asn--Leu sequences                (B) LOCATION: bp 2932, 3160, 3355, 3583, 4150, 4444, 4672,                    4900, 5128, 5356, 5584:                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: dyad symmetry regions                                           (B) LOCATION: from bp 6554 to 6566 and from bp 6571 to 6583                   (ix) FEATURE:                                                                 (A) NAME/KEY: dyad symmetry regions                                           (B) LOCATION: from bp 6611 to 6625 and from bp 6631 to 6644                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TTGAACAACTTAAAACTAGTTAGTTTTGTTTAAAATGTAATTGAATTGTCTTTTTAAGTA60                GGCTGTTTACACGATATTTGTCTTCCTTTATATAAATATGATAGATTTTCAGTAAATTTT120               TCAAAAAAACCTCAAAAATAACAGATTTTTTCTTGTATCTTTGAGGCATAAGGAGTATAA180               TGGTGACGGTATTCAAGTAGAAATTTTATATACTCTTGATGAAAACATTCTGTCTACTTT240               AAAATAAATAATCTACTGGGTATCCTTCTGCTAAGTTTTTAAAGCAGGAGGTGTGTTTTT300               GTACATGGTGTTACAGGAACCAGAAATGATCGATTCGCCAGTAAAATATAGGAGGATATC360               ATGTCTTATAAAGATATGTTCAGAAAAGAACAACGTTTTTCTTTTCGT408                           MetSerTyrLysAspMetPheArgLysGluGlnArgPheSerPheArg                              45-40-35                                                                      AAATTTAGCTTTGGTCTAGCTTCGGCAGTCATTGCAAACGTTATTTTG456                           LysPheSerPheGlyLeuAlaSerAlaValIleAlaAsnValIleLeu                              30-25-20-15                                                                   GGAGGAGCAATCGCAAACAGCCCTGTTGTTCATGCTAACACAGTGACA504                           GlyGlyAlaIleAlaAsnSerProValValHisAlaAsnThrValThr                              10-51                                                                         GAAGCAGAGACAGCTGTAGCACCAGCTAACCAAGACCTTGGAAATGAG552                           GluAlaGluThrAlaValAlaProAlaAsnGlnAspLeuGlyAsnGlu                              51015                                                                         ACTAAAACGGAAGAAGAACCCAAGGAACCAATCGAAGCAGTTCGCACG600                           ThrLysThrGluGluGluProLysGluProIleGluAlaValArgThr                              202530                                                                        GACATGGAAAACCGTGCAGCTGAAATCTTGCCGGAGGCGCTGAATGCT648                           AspMetGluAsnArgAlaAlaGluIleLeuProGluAlaLeuAsnAla                              35404550                                                                      AGTGTAACAAACCAAGCACCAGTTATTCCGACTATTGGAGATCTTCCT696                           SerValThrAsnGlnAlaProValIleProThrIleGlyAspLeuPro                              556065                                                                        AAAGATGCGAGTGGTCAGAATGTTCATGGTAAGGCAACGGATAATAAG744                           LysAspAlaSerGlyGlnAsnValHisGlyLysAlaThrAspAsnLys                              707580                                                                        ATTTATCGTGTTGTATACGTTTTTGGTAATGTAGCAGGGACTACGGAG792                           IleTyrArgValValTyrValPheGlyAsnValAlaGlyThrThrGlu                              859095                                                                        ACAGAAGATGGTAAACAAAATGTTGCTCCAACATTTAACAGAAATGAT840                           ThrGluAspGlyLysGlnAsnValAlaProThrPheAsnArgAsnAsp                              100105110                                                                     GCAACTAAAACTTTTCCAATCACAGATCCAGATAGCGACATTCAAACT888                           AlaThrLysThrPheProIleThrAspProAspSerAspIleGlnThr                              115120125130                                                                  ATTTCATACGAAGTTCCAGCTGATATTGCAAGCTATACCTTGGATGAT936                           IleSerTyrGluValProAlaAspIleAlaSerTyrThrLeuAspAsp                              135140145                                                                     CCAAACTCAATTGTTACTAATGGCACCTCACCTGGTCCAGTATCTTAC984                           ProAsnSerIleValThrAsnGlyThrSerProGlyProValSerTyr                              150155160                                                                     TTAGATGGTCCAAATGGGTCAGCCACTCTCACACAAGATGGTTATCTA1032                          LeuAspGlyProAsnGlySerAlaThrLeuThrGlnAspGlyTyrLeu                              165170175                                                                     ACAGGAAGTTTCCCTTGGGGAGCAGGAGACCTAGCTGGTCGTCGGATT1080                          ThrGlySerPheProTrpGlyAlaGlyAspLeuAlaGlyArgArgIle                              180185190                                                                     AAAGTGACGGATGCCACTGGTAATACTACTAAGAGTAATCCGTTCTAT1128                          LysValThrAspAlaThrGlyAsnThrThrLysSerAsnProPheTyr                              195200205210                                                                  ATGGTTGCATATACAGTCAAGCCAGTAGATGATAAACCTCTAGCAGTA1176                          MetValAlaTyrThrValLysProValAspAspLysProLeuAlaVal                              215220225                                                                     TCAAACTCTTCTGAGCTGACGGAACAGGCTATTTTTGATAAGTTGGTT1224                          SerAsnSerSerGluLeuThrGluGlnAlaIlePheAspLysLeuVal                              230235240                                                                     GTCGATAAGTCTGCTAAAACAACTTCAAATAGCGCTCTTGTAATTGAT1272                          ValAspLysSerAlaLysThrThrSerAsnSerAlaLeuValIleAsp                              245250255                                                                     TCTAGCAACTACAAACATTCAATTGCAGGTTATCGTACCGTAAATTCT1320                          SerSerAsnTyrLysHisSerIleAlaGlyTyrArgThrValAsnSer                              260265270                                                                     GATGGCACAAAAACAGAAACAGTAGAGGAAACAAATCTATCTGATTTC1368                          AspGlyThrLysThrGluThrValGluGluThrAsnLeuSerAspPhe                              275280285290                                                                  CCAACTGAAGGTAAATACGAAGTTCGAGTAAAAACAACCAATGTTTAC1416                          ProThrGluGlyLysTyrGluValArgValLysThrThrAsnValTyr                              295300305                                                                     GGTCAAACTATCTACAACTGGATTCCTGTAAATGCCTATAAGTTGGAC1464                          GlyGlnThrIleTyrAsnTrpIleProValAsnAlaTyrLysLeuAsp                              310315320                                                                     ACAGCGAAGGATGCTGAAATTCGGAAGTATACAGACAACCAAGCCCCA1512                          ThrAlaLysAspAlaGluIleArgLysTyrThrAspAsnGlnAlaPro                              325330335                                                                     ATTCATGCTATAATGCAAATTGGTCAAGCTGGAGAAAAGGCAGCAGTT1560                          IleHisAlaIleMetGlnIleGlyGlnAlaGlyGluLysAlaAlaVal                              340345350                                                                     ATATTGAAGGATATTCCATCCGATTTCAGTATTGAAAACTTCAATTTG1608                          IleLeuLysAspIleProSerAspPheSerIleGluAsnPheAsnLeu                              355360365370                                                                  AAAGATGGTGTAGCAGATGAGCTTGCTAAACGTAACTTGGAATTTGTA1656                          LysAspGlyValAlaAspGluLeuAlaLysArgAsnLeuGluPheVal                              375380385                                                                     AGAAATGATGCAGTGGCGACAACTGATACTGATGGAGATGGCGCCAAA1704                          ArgAsnAspAlaValAlaThrThrAspThrAspGlyAspGlyAlaLys                              390395400                                                                     GAAGGAATTGTTGGATATATTCAACCAAAAACTGGCGGTGCAAACAGT1752                          GluGlyIleValGlyTyrIleGlnProLysThrGlyGlyAlaAsnSer                              405410415                                                                     GGGGTAGCCACTTATACAGGATCAAATAATCTTACTTATGGCTTCACT1800                          GlyValAlaThrTyrThrGlySerAsnAsnLeuThrTyrGlyPheThr                              420425430                                                                     TACAAAGCTGTTGAGACAAAAGATAAGGCGAATGCCACAGAGGCTAAA1848                          TyrLysAlaValGluThrLysAspLysAlaAsnAlaThrGluAlaLys                              435440445450                                                                  ACTCTCGAATTAGATTACACCATCTTATTCATAGATACTAAAGCACCA1896                          ThrLeuGluLeuAspTyrThrIleLeuPheIleAspThrLysAlaPro                              455460465                                                                     GTCATGACACCTAAATCAGAGTACATCCGTTTTGTTGGTGAAGAGTAT1944                          ValMetThrProLysSerGluTyrIleArgPheValGlyGluGluTyr                              470475480                                                                     ACGGTTAGCGTCCCAGGTACGGATAACGCCTTCCTTAATACCGGCAAA1992                          ThrValSerValProGlyThrAspAsnAlaPheLeuAsnThrGlyLys                              485490495                                                                     CTAAATGGAACTCTCTCAATTTTGAAAGATGGAGAGTCAGGTTCTCTT2040                          LeuAsnGlyThrLeuSerIleLeuLysAspGlyGluSerGlySerLeu                              500505510                                                                     GTATCATCAGACTTAGGTACAAACACTAAGATTACTTCAGAACTGGAT2088                          ValSerSerAspLeuGlyThrAsnThrLysIleThrSerGluLeuAsp                              515520525530                                                                  CCTACGGGAGCAACTGCAAACCAAGGAGATGACGGTCAATCTTCAACT2136                          ProThrGlyAlaThrAlaAsnGlnGlyAspAspGlyGlnSerSerThr                              535540545                                                                     AAGTTTAACGTTAAGATTACAGGTACCGGACCTGCTACAGAAGGTACC2184                          LysPheAsnValLysIleThrGlyThrGlyProAlaThrGluGlyThr                              550555560                                                                     GGCACTTATAAGCTTCGTGTTGGAGAAGATAACTATCCTTTTGGTCCA2232                          GlyThrTyrLysLeuArgValGlyGluAspAsnTyrProPheGlyPro                              565570575                                                                     GAGGGGAAACTTGTTGATGGAAATAAACCAGAAAATGTAGGTTTGACA2280                          GluGlyLysLeuValAspGlyAsnLysProGluAsnValGlyLeuThr                              580585590                                                                     TCTGTAAAAGTTACCTTCGTAAAACATGCTACGGTGTCAACACCAGTT2328                          SerValLysValThrPheValLysHisAlaThrValSerThrProVal                              595600605610                                                                  TCTGTTGAAAATCCAGCTAACTTAACGCCAGAAGAAAAAGCCGCAGTT2376                          SerValGluAsnProAlaAsnLeuThrProGluGluLysAlaAlaVal                              615620625                                                                     ATTGCTCAAATCAAGAAAGACAACGCAGACAACGAAAGATTGAAGGGC2424                          IleAlaGlnIleLysLysAspAsnAlaAspAsnGluArgLeuLysGly                              630635640                                                                     TTGCCAGATTCAGCATTTACAGTTAACTCAGATGGTACTGTGTCAGTT2472                          LeuProAspSerAlaPheThrValAsnSerAspGlyThrValSerVal                              645650655                                                                     GACTACAGTGCCGGTGGTGTCAATGTTGATGGTGCGACAGACATTATT2520                          AspTyrSerAlaGlyGlyValAsnValAspGlyAlaThrAspIleIle                              660665670                                                                     AAGAATGCTACCACAAACTTGGCAGATACACGGAATGAAGCAAAAGCA2568                          LysAsnAlaThrThrAsnLeuAlaAspThrArgAsnGluAlaLysAla                              675680685690                                                                  GAAATCGACACAAAATTAGCTGAACATAAAAAAGCTATCGAAGCAAAA2616                          GluIleAspThrLysLeuAlaGluHisLysLysAlaIleGluAlaLys                              695700705                                                                     CGGGATGAAGCGTTTTCTAAAATTGATGATGACATTTCCTTGAGAGCA2664                          ArgAspGluAlaPheSerLysIleAspAspAspIleSerLeuArgAla                              710715720                                                                     GAACAGAGACAGGCTGCTAAGGATGCCGTTGCTGCAGCTGCTGGGGAT2712                          GluGlnArgGlnAlaAlaLysAspAlaValAlaAlaAlaAlaGlyAsp                              725730735                                                                     GCTTTGAAAGAATTAGACAACAAGGCGACAGAAGCAAAAGAAAAAATT2760                          AlaLeuLysGluLeuAspAsnLysAlaThrGluAlaLysGluLysIle                              740745750                                                                     GATAAAGCTACGACGGCCTCAGAAATCAATGATGCTAAGACTAATGGT2808                          AspLysAlaThrThrAlaSerGluIleAsnAspAlaLysThrAsnGly                              755760765770                                                                  GAGATTAATCTGGACAGTGCAGAAGCAGTAGGCGAAAAAGCTATTAAC2856                          GluIleAsnLeuAspSerAlaGluAlaValGlyGluLysAlaIleAsn                              775780785                                                                     CAGGCGAAGGAAAAAGAACTGGCAAAAGCAGAAGTTGAAAACAAAGCA2904                          GlnAlaLysGluLysGluLeuAlaLysAlaGluValGluAsnLysAla                              790795800                                                                     TTCGAGGCATTGGAAAAAGTTAACAATAATCCAAACTTGTTAGAAGAA2952                          PheGluAlaLeuGluLysValAsnAsnAsnProAsnLeuLeuGluGlu                              805810815                                                                     GAGAAAAAAGCATACTTTGATGATATTAAAGAATCTAAAGAAGTTGCA3000                          GluLysLysAlaTyrPheAspAspIleLysGluSerLysGluValAla                              820825830                                                                     GTTGAGAAAATCAATAATGCTGAAAATACTGCTGAAATTACGGCAGCA3048                          ValGluLysIleAsnAsnAlaGluAsnThrAlaGluIleThrAlaAla                              835840845850                                                                  ATTGACGAAGCGGAAATTGCATACAATGAAGATGTTATTAACGCAGCC3096                          IleAspGluAlaGluIleAlaTyrAsnGluAspValIleAsnAlaAla                              855860865                                                                     CAACTTGATGCTTTGAATAAGCTTGAAAAAGATAGCGAAGAAACTAAG3144                          GlnLeuAspAlaLeuAsnLysLeuGluLysAspSerGluGluThrLys                              870875880                                                                     GCAGCTATTGATGCTAATCCAAACTTAACTCCGGAAGAGAAAGCGAAA3192                          AlaAlaIleAspAlaAsnProAsnLeuThrProGluGluLysAlaLys                              885890895                                                                     GCTATTGCTAAGGTAGAAGAGCTTGTTAATAATGCTGAATCTGACATT3240                          AlaIleAlaLysValGluGluLeuValAsnAsnAlaGluSerAspIle                              900905910                                                                     TTGTCGAAGCCTACCCCAGAAACAGTTCAAGCAGTGGAGGATAAGGCT3288                          LeuSerLysProThrProGluThrValGlnAlaValGluAspLysAla                              915920925930                                                                  GACAAAGATCTTGCCAAAGTAGAACTTCAAGCAGCAGCAGACGGTGCG3336                          AspLysAspLeuAlaLysValGluLeuGlnAlaAlaAlaAspGlyAla                              935940945                                                                     AAGAAAGGCATTGAAGCAAATCCGAATTTGACTCCAGAAGAGAAAGAT3384                          LysLysGlyIleGluAlaAsnProAsnLeuThrProGluGluLysAsp                              950955960                                                                     GTAGCTAAGAAGGCAGTAGAAGACGCGGTTAAGGTGGCGACAGACGCT3432                          ValAlaLysLysAlaValGluAspAlaValLysValAlaThrAspAla                              965970975                                                                     ATTGATAAGGCGTCAACTCCAACCGAAGTTGACACAGCGACAAGCGAT3480                          IleAspLysAlaSerThrProThrGluValAspThrAlaThrSerAsp                              980985990                                                                     GGAGTGAAGGCTATTGATGCAGAAGAGTTTAAAGCTACTCAGAAAGAT3528                          GlyValLysAlaIleAspAlaGluGluPheLysAlaThrGlnLysAsp                              995100010051010                                                               GCTAAGAACAAGATTGCCAAAGAAGCAGAATCAGCTAAGAAAGCGATT3576                          AlaLysAsnLysIleAlaLysGluAlaGluSerAlaLysLysAlaIle                              101510201025                                                                  GACGACAATCCAAACTTGACTCCAGATGAGAAGGAATCAGCTAAGAAT3624                          AspAspAsnProAsnLeuThrProAspGluLysGluSerAlaLysAsn                              103010351040                                                                  GCAGTGGAAGAGGCGGCTAAGGTAGCAACAGCCGCTATTGATAAAGCA3672                          AlaSerGluGluAlaAlaLysValAlaThrAlaAlaIleAspLysAla                              104510501055                                                                  TCAACTCCAGATGCAGTTCAAGTAGAAGAGGACAAAGGTGTAGCAGCT3720                          SerThrProAspAlaValGlnValGluGluAspLysGlyValAlaAla                              106010651070                                                                  ATCAATTTGATTACTGCCAAGGCAGATGCTAAAGGTGTCATTGCTGCT3768                          IleAsnLeuIleThrAlaLysAlaAspAlaLysGlyValIleAlaAla                              1075108010851090                                                              AAGTTGGCAGATGAAATCAAGAAGCTCGAAGATAAGCAAGCAGAAGCA3816                          LysLeuAlaAspGluIleLysLysLeuGluAspLysGlnAlaGluAla                              109511001105                                                                  GAAAAAGCTATCGATGCGTCAACTATGACTAATGAGGAGAAAGCAATC3864                          GluLysAlaIleAspAlaSerThrMetThrAsnGluGluLysAlaIle                              111011151120                                                                  GCTAAGAAGGCTCTTCAAGATGTTGTAGATAAAGGAAAAGCAGAGCTT3912                          AlaLysLysAlaLeuGlnAspValValAspLysGlyLysAlaGluLeu                              112511351135                                                                  GAAGACGCAGCTAGGGTAGCAACAAATGAGATTCATGAAGCTACTACT3960                          GluAspAlaAlaArgValAlaThrAsnGluIleHisGluAlaThrThr                              114011451150                                                                  ACAGAAAAAGCGAAAGCGGCGGAACTTGCTGGCGAAAAGAGCTTGACA4008                          ThrGluLysAlaLysAlaAlaGluLeuAlaGlyGluLysSerLeuThr                              1155116511651170                                                              GACACAGGTAAAGAAGCTAGAGATGCAGTTGAATTGGCTAAGGATAAA4056                          AspThrGlyLysGluAlaArgAspAlaValGluLeuAlaLysAspLys                              117511801185                                                                  GAATTAGCTAAGGAAGCAATCCGAACAGAAGAAGAAGAAGCTACTAAA4104                          GluLeuAlaLysGluAlaIleArgThrGluGluGluGluAlaThrLys                              119011951200                                                                  ATAGTAGAGAAACTTGCAGAAGATACGCGCAAAGCTATCGAGGACAAT4152                          IleValGluLysLeuAlaGluAspThrArgLysAlaIleGluAspAsn                              120512101215                                                                  CCAAACTTGTCAGATGAAGATAAGCAAGCGGAAATTAAAAAGCTAACT4200                          ProAsnLeuSerAspGluAspLysGlnAlaGluIleLysLysLeuThr                              122012251230                                                                  GACGCTGTGGCAAAAACTTTAGCAACCATTCGTGACAATGCAGATAAG4248                          AspAlaValAlaLysThrLeuAlaThrIleArgAspAsnAlaAspLys                              1235124012451250                                                              CGTACGCAAGAAGCAGAAAAAGCTCAAGCCCTAGCAGATCTTGAAAAA4296                          ArgThrGlnGluAlaGluLysAlaGlnAlaLeuAlaAspLeuGluLys                              125512601265                                                                  GCTAAAGAAACACAGAAAATTGCAGATAAAGCTGCGATTGATAGGTTG4344                          AlaLysGluThrGlnLysIleAlaAspLysAlaAlaIleAspArgLeu                              127012751280                                                                  ACTATACTTGTGAAAGATGGTGAGCTTGAAGCTACTAAACAAGATGCT4392                          ThrIleLeuValLysAspGlyGluLeuGluAlaThrLysGlnAspAla                              128512901295                                                                  AAGAACAAGATTGCTAAAGATGCAGCCGCTGCTAAAGAAGCAATTGCA4440                          LysAsnLysIleAlaLysAspAlaAlaAlaAlaLysGluAlaIleAla                              130013051310                                                                  AGCAATCCAAACTTGACAGACGCAGAGAAGAAAACCTTCACCGATGCG4488                          SerAsnProAsnLeuThrAspAlaGluLysLysThrPheThrAspAla                              1315132013251330                                                              GTAGATGCAGAAGTAGCCAAAGCTAACGACGCAATTTCAGCTGCAACC4536                          ValAspAlaGluValAlaLysAlaAsnAspAlaIleSerAlaAlaThr                              133513401345                                                                  AGCCCAGCAGATGTTCAAAAAGAAGAGGATGCAGGTGTTGCAGCCATT4584                          SerProAlaAspValGlnLysGluGluAspAlaGlyValAlaAlaIle                              135013551360                                                                  GCAGAAGATGTTCTTGACGCAGCTAAACAAGATGCTAAGAATAAGATT4632                          AlaGluAspValLeuAspAlaAlaLysGlnAspAlaLysAsnLysIle                              136513701375                                                                  GCTAAAGATGCAGCCGCTGCTAAAGAAGCAATTGGCTCCAATCCAAAC4680                          AlaLysAspAlaAlaAlaAlaLysGluAlaIleGlySerAsnProAsn                              138013851390                                                                  TTGACAGACGCAGAGAAGAAAACCTTCACCGATGCGGTAGATGCAGAA4728                          LeuThrAspAlaGluLysLysThrPheThrAspAlaValAspAlaGlu                              1395140014051410                                                              GTAGCCAAAGCTAACGACGCAATTTCAGCTGCAACCAGCCCAGCAGAT4776                          ValAlaLysAlaAsnAspAlaIleSerAlaAlaThrSerProAlaAsp                              141514201425                                                                  GTTCAAAAAGAAGAGGATGCAGGTGTTGCAGCCATTGCAGAAGATGTT4824                          ValGlnLysGluGluAspAlaGlyValAlaAlaIleAlaGluAspVal                              143014351440                                                                  CTTGACGCAGCTAAACAAGATGCTAAGAATAAGATTGCTAAAGAATCC4872                          LeuAspAlaAlaLysGlnAspAlaLysAsnLysIleAlaLysGluSer                              144514501455                                                                  GACGCTGCTAAGTCAGCCATTGACGCGAATCCAAACTTGACAGATGCA4920                          AspAlaAlaLysSerAlaIleAspAlaAsnProAsnLeuThrAspAla                              146014651470                                                                  GAGAAGGAATCAGCTAAGAAAGCAGTTGATGCTGATGCTAAAGCTGCG4968                          GluLysGluSerAlaLysLysAlaValAspAlaAspAlaLysAlaAla                              1475148014851490                                                              ACAGATGCAATTGATGCTTCAACAAGTCCAGTCGAAGCGCAATCGGCA5016                          ThrAspAlaIleAspAlaSerThrSerProValGluAlaGlnSerAla                              149515001505                                                                  GAGGACAAAGGCGTAGGTTCAATCGCCCAAGATGTTCTTGACGCAGCG5064                          GluAspLysGlyValGlySerIleAlaGlnAspValLeuAspAlaAla                              151015151520                                                                  AAACAAGATGCTAAGAACAAGATTGCCAAAGAAGTTGCCGCAGCTAAA5112                          LysGlnAspAlaLysAsnLysIleAlaLysGluValAlaAlaAlaLys                              152515301535                                                                  GAAGCAATTGATGCCAATCCGAACTTATCAGATGCAGAGAAGGAAGCT5160                          GluAlaIleAspAlaAsnProAsnLeuSerAspAlaGluLysGluAla                              154015451550                                                                  TCTAAGAAAGCGGTAGATGCAGATGCTAAAGCTACGACAGATGCAATT5208                          SerLysLysAlaValAspAlaAspAlaLysAlaThrThrAspAlaIle                              1555156015651570                                                              GATGCTTCAACAAGTCCAGTCGAAGCGCAATCGGCAGAGGACAAAGGC5256                          AspAlaSerThrSerProValGluAlaGlnSerAlaGluAspLysGly                              157515801585                                                                  GTAGGTTCAATCGCCCAAGATGTTCTTGACGCAGCGAAACAAGATGCT5304                          ValGlySerIleArgGlnAspValLeuAspAlaAlaLysGlnAspAla                              159015951600                                                                  AAGAATAAGATTGCTAAAGAATCCGACGCTGCTAAGTCAGCCATTGAC5352                          LysAsnLysIleAlaLysGluSerAspAlaAlaLysSerAlaIleAsp                              160516101615                                                                  GCGAATCCAAACTTGACAGATGCAGAGAAGGAATCAGCTAAGAAAGCG5400                          AlaAsnProAsnLeuThrAspAlaGluLysGluSerAlaLysLysAla                              162016251630                                                                  GTAGATGCAGATGCTAAAGCTGCGACAGATGCAATTGATGCTTCAACA5448                          ValAspAlaAspAlaLysAlaAlaThrAspAlaIleAspAlaSerThr                              1635164016451650                                                              AGTCCAGTCGAAGCGCAATCGGCAGAGGACAAAGGCGTAGGCGCCATC5496                          SerProValGluAlaGlnSerAlaGluAspLysGlyValGlyAlaIle                              165516601665                                                                  GCCAAAGACATTCTTGATGCCGCGAAACAAGATGCTAAGAACAAGATT5544                          AlaLysAspIleLeuAspAlaAlaLysGlnAspAlaLysAsnLysIle                              167016751680                                                                  GCTAAAGAGGCAGAATCCGCTAAGTCAGTCATTGACTCCAATCCGAAC5592                          AlaLysGluAlaGluSerAlaLysSerValIleAspSerAsnProAsn                              168516901695                                                                  TTGACAGATGCAGCTAAGGAAGCGGCTAAATCTGAAATTGATAAAGCT5640                          LeuThrAspAlaAlaLysGluAlaAlaLysSerGluIleAspLysAla                              170017051710                                                                  GTTGAGGAAGCGATTGTTTTAATCAATGGTGTTAGAACTTATCAAGAG5688                          ValGluGluAlaIleValLeuIleAsnGlyValArgThrTyrGlnGlu                              1715172017251730                                                              TTGGAAAAAATCAAACTTCCAATGGCAGCTCTAATTAAACCAGCTGCG5736                          LeuGluLysIleLysLeuProMetAlaAlaLeuIleLysProAlaAla                              173517401745                                                                  AAAGTAACACCAGTGGTTGATCCAAATAACTTGACTGAAAAAGAAATT5784                          LysValThrProValValAspProAsnAsnLeuThrGluLysGluIle                              175017551760                                                                  GCTCGTATCAAGGCATTCCTTAAAGAGAACAATAACCTCCCATAA5829                             AlaArgIleLysAlaPheLeuLysGluAsnAsnAsnLeuPro                                    176517701775                                                                  GGAACAGAGATTAATGTTTCTAAAGATGCTTCAGTGACAATTAAATATCCAGATGGAACT5889              ATTGATTTGCTATCACCAGTAGAAGTTGTGAAGCAGGCAGATAAAACTGCTCCTACGGTC5949              GCAAATGATGGCAAAGGTAATATTGTGATTGTACCGTCTGAAAAAGCTGTTGAGCTTGTT6009              GTTTCATACGTAGATAACAATGGTAAGTCGCAAACTGTAGTTGTTACGAAAGGTACGGAT6069              GGTTTATGGACAGCAAGTAATACAGTGGTGATTGTGGACCCTGTGACTGGGCAAGTAATC6129              GTTCCAGGTTCTGTTATTAAGCCAGGTACAGTTGTTACAGCATACTCTAAAGACGAGGTT6189              GGAAATAGTTCTGATTCAGCAGAAGCTGAAGTTGTAGCAGTAGACGAAAATAATTCTGCA6249              GCAGGAGTGAAAGTTAAATCAGTTACTACAAATGCTAATAATGTTGAGAAGAAAGCTAAG6309              CAATTACCGAATACTGGTGAGGAAGCAAATTCAGCAACTTCACTCGGATTAGTAGCTCTT6369              GGACTCGGATTAGCACTTCTTGCAGCAAAGAGAAGAAGAGACGAAGAAGCTTAAGATAAG6429              CTCTTCCTCAGAACTCTTTTGGAAGCCGCAATTTTCCTAGAAGATAGTAGTATGATACTC6489              TTTCATAGCAAGGAAATTCCCTCGCTATGATTGGTAGGTATCAGTTATTATCTATCGAAC6549              CCCCAAAATCCAAAGTCATTCGACTTTGGATTTTTTTGATACGACATGCTCGTCATACCT6609              AAAAAACAGCCTTCTCTTGCCGAGAGGCTGTTTTTCATGCTTTTAATCTAAAAGTCTGCG6669              GACGTTTTTTCAATAAAATCCAGTAACCGATGCTAACATAGGCAATCATAGCTAGGGAAA6729              CCAGCAGGATATAGG6744                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4118 base pairs                                                   (B) TYPE: Nucleic acid with corresponding amino acids                         (C) STRANDEDNESS: single stranded                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: genomic DNA                                               (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Streptococcus suis type II (pathogenic)                         (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Muramidase released protein (MRP) gene                 (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 4 to 9                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 29 to 34                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -35 region                                             (B) LOCATION: bp 40 to 45                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: promoter -10 region                                             (B) LOCATION: bp 63 to 68                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: ribosome binding site                                           (B) LOCATION: bp 147 to 152                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: signal peptide                                                  (B) LOCATION: bp 159 to 299                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: mature peptide                                                  (B) LOCATION: bp 300 to 3926                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: proline rich region                                             (B) LOCATION: from bp 2757 to 3014                                            (ix) FEATURE:                                                                 (A) NAME/KEY: repetitive units                                                (B) LOCATION: from bp 3015 to 3176, 3423 to 3584 and 3585 to                  3743                                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: membrane anchor sequence                                        (B) LOCATION: from bp 3825 to 3926                                            (ix) FEATURE:                                                                 (A) NAME/KEY: dyad symmetry regions                                           (B) LOCATION: from bp 4069 to 4080 and from bp 4087 to 4098                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAATTCATAATGTTTTTTTGAGGAATTTTATAATATTACTTGGCATTTAAAGTTATTTGT60                AGTATAATACCTCGAATGATTGCGGGAGTTTTCAAGGCTTTGATACAAAGAGTAGAAAAT120               TTGTGTAATTAAATTAATATTTATATGGGGGATTTTTT158                                     ATGCGTAGATCAAATAAAAAATCATTTGACTGGTACGGTACGAAACAA206                           MetArgArgSerAsnLysLysSerPheAspTrpTyrGlyThrLysGln                              45-40-35                                                                      CAATTTTCGATTCGTAAGTATCATTTTGGGGCAGCAAGCGTTTTGCTT254                           GlnPheSerIleArgLysTyrHisPheGlyAlaAlaSerValLeuLeu                              30-25-20                                                                      GGTGTGTCGTTAGTTTTAGGTGCTGGTGCACAGGTTGTTAAGGCTGAT302                           GlyValSerLeuValLeuGlyAlaGlyAlaGlnValValLysAlaAsp                              15-10-51                                                                      GAAACTGTTGCTTCATCAGAACCAACTATTGCCAGTAGTGTAGCGCCT350                           GluThrValAlaSerSerGluProThrIleAlaSerSerValAlaPro                              51015                                                                         GCTTCAACAGAAGCGGTTGCAGAAGAAGCAGAAAAAACAAATGCTGAA398                           AlaSerThrGluAlaValAlaGluGluAlaGluLysThrAsnAlaGlu                              202530                                                                        AATACGAGTGCAGTAGCTACGACTTCAACAGAAGTTGAAAAAGCGAAA446                           AsnThrSerAlaValAlaThrThrSerThrGluValGluLysAlaLys                              354045                                                                        GCTGTTCTTGAACAGGTAACATCAGAATCACCACTTTTGGCTGGTCTT494                           AlaValLeuGluGlnValThrSerGluSerProLeuLeuAlaGlyLeu                              50556065                                                                      GGTCAAAAAGAGTTGGCTAAAACTGAAGATGCAACTCTTGCAAAAGCT542                           GlyGlnLysGluLeuAlaLysThrGluAspAlaThrLeuAlaLysAla                              707580                                                                        ATAGAGGATGCTCAAACAAAACTTGCAGCAGCTAAGGCAATTTTGGCT590                           IleGluAspAlaGlnThrLysLeuAlaAlaAlaLysAlaIleLeuAla                              859095                                                                        GACTCAGAAGCAACTGTTGAGCAAGTTGAAGCGCAAGTCGCAGCGGTT638                           AspSerGluAlaThrValGluGlnValGluAlaGlnValAlaAlaVal                              100105110                                                                     AAAGTAGCCAACGAGGCGCTAGGGAATGAATTGCAAAAATACACTGTA686                           LysValAlaAsnGluAlaLeuGlyAsnGluLeuGlnLysTyrThrVal                              115120125                                                                     GATGGTCTCTTGACAGCGGCTCTTGATACAGTAGCACCTGATACAACT734                           AspGlyLeuLeuThrAlaAlaLeuAspThrValAlaProAspThrThr                              130135140145                                                                  GCATCAACATTGAAAGTTGGTGATGGCGAAGGTACCCTTCTAGATAGC782                           AlaSerThrLeuLysValGlyAspGlyGluGlyThrLeuLeuAspSer                              150155160                                                                     ACTACAACAGCAACGCCTTCAATGGCTGAGCCAAATGGTGCAGCAATT830                           ThrThrThrAlaThrProSerMetAlaGluProAsnGlyAlaAlaIle                              165170175                                                                     GCTCCACATACACTTCGAACTCAAGATGGAATTAAAGCGACATCAGAG878                           AlaProHisThrLeuArgThrGlnAspGlyIleLysAlaThrSerGlu                              180185190                                                                     CCAAATTGGTATACTTTTGAATCGTACGATTTGTACTCATATAATAAA926                           ProAsnTrpTyrThrPheGluSerTyrAspLeuTyrSerTyrAsnLys                              195200205                                                                     AATATGGCTAGCTCAACTTATAAAGGAGCTGAAGTTGATGCCTACATT974                           AsnMetAlaSerSerThrTyrLysGlyAlaGluValAspAlaTyrIle                              210215220225                                                                  CGTTACTCTTTGGATAATGATTCGTCAACAACTGCTGTTTTAGCAGAG1022                          ArgTyrSerLeuAspAsnAspSerSerThrThrAlaValLeuAlaGlu                              230235240                                                                     TTGGTAAGTAGGACAACTGGTGATGTGTTAGAGAAATATACGATTGAA1070                          LeuValSerArgThrThrGlyAspValLeuGluLysTyrThrIleGlu                              245250255                                                                     CCGGGCGAGAGTGTTACGTTTTCACATCCGACAAAAGTTAATGCTAAT1118                          ProGlyGluSerValThrPheSerHisProThrLysValAsnAlaAsn                              260265270                                                                     AATAGCAATATAACTGTGACTTATGATACCTCATTAGCTTCTGCTAAT1166                          AsnSerAsnIleThrValThrTyrAspThrSerLeuAlaSerAlaAsn                              275280285                                                                     ACTCCTGGAGCATTGAAATTCTCTGCTAATGATGATGTTTATTCAACA1214                          ThrProGlyAlaLeuLysPheSerAlaAsnAspAspValTyrSerThr                              290295300305                                                                  ATTATTGTACCTGCTTATCAGATTAATACAACTCGTTACGTCACTGAA1262                          IleIleValProAlaTyrGlnIleAsnThrThrArgTyrValThrGlu                              310315320                                                                     AGTGGCAAAGTTTTGGCAACCTATGGTCTTCAAACTATTGCAGGACAG1310                          SerGlyLysValLeuAlaThrTyrGlyLeuGlnThrIleAlaGlyGln                              325330335                                                                     GTAGTTACTCCATCTTCTGTTCGTGTATTTACTGGGTATGATTATGTG1358                          ValValThrProSerSerValArgValPheThrGlyTyrAspTyrVal                              340345350                                                                     GCAACTACAACTAAAGCCGTTCAAGGTCCATATCCAAAGGGAACGGTA1406                          AlaThrThrThrLysAlaValGlnGlyProTyrProLysGlyThrVal                              355360365                                                                     TACCTTGCTGGTACGGTTCAAAAGGATACAGTACAATATAAAGTTATT1454                          TyrLeuAlaGlyThrValGlnLysAspThrValGlnTyrLysValIle                              370375380385                                                                  CGTGAAATTGTGGAGAACGACCAAGCAGTTCTTAAATTCTATTATTTA1502                          ArgGluIleValGluAsnAspGlnAlaValLeuLysPheTyrTyrLeu                              390395400                                                                     GATCCTACCTATAAGGGTGAAGTAGATTGGAGAGGAACTGATACGACT1550                          AspProThrTyrLysGlyGluValAspTrpArgGlyThrAspThrThr                              405410415                                                                     GGGTTTATTGAGTTGCTTACAACTTCCCCAACAACCTATAAAGTTGGT1598                          GlyPheIleGluLeuLeuThrThrSerProThrThrTyrLysValGly                              420425430                                                                     ACTATATACGATTACAATATTAATTCAAAAATTACAGCTCCATTTACT1646                          ThrIleTyrAspTyrAsnIleAsnSerLysIleThrAlaProPheThr                              435440445                                                                     ATTGATCCTACCAAGAATGTTATGGTTTTCAAGGAAAGTGAACAGAAC1694                          IleAspProThrLysAsnValMetValPheLysGluSerGluGlnAsn                              450455460465                                                                  GAGCAAGGTAGCAAATATCGCGTCATTGCTCAATGGTCAGGAGATGAA1742                          GluGlnGlySerLysTyrArgValIleAlaGlnTrpSerGlyAspGlu                              470475480                                                                     ACCACTAAAGGTATATATGGAAAAATCTATATCGCTACTCAGGTTTGG1790                          ThrThrLysGlyIleTyrGlyLysIleTyrIleAlaThrGlnValTrp                              485490495                                                                     ACGACTAAATTGGGAACAAACGAGTGGGGATGGTTTGACTATTCTGAT1838                          ThrThrLysLeuGlyThrAsnGluTrpGlyTrpPheAspTyrSerAsp                              500505510                                                                     GACCAAGCTGGTATAAAATTTAATAACAAAGGTTTTTGGCCGGCAGGT1886                          AspGlnAlaGlyIleLysPheAsnAsnLysGlyPheTrpProAlaGly                              515520525                                                                     GTTCAAAATACACTTCGAAATGCTACTCCAGCTACAGCTGTAGAGACT1934                          ValGlnAsnThrLeuArgAsnAlaThrProAlaThrAlaValGluThr                              530535540545                                                                  ACTTATATCTACAAAGAAAGTTCCAAGTATGGTGATGTCATTGTTGAG1982                          ThrTyrIleTyrLysGluSerSerLysTyrGlyAspValIleValGlu                              550555560                                                                     TACTACGATACTGACGGAAAACAAATTGTAAATTCAGTTGTAGATACT2030                          TyrTyrAspThrAspGlyLysGlnIleValAsnSerValValAspThr                              565570575                                                                     CCTAAGTCAGCTCTTGGCACAGAGTATAATACAGATGTGGACCGTAGA2078                          ProLysSerAlaLeuGlyThrGluTyrAsnThrAspValAspArgArg                              580585590                                                                     CCAGCCAGCTTGGTTGCTGCTGATGGGACAGTCTACTTCTACAAAGAA2126                          ProAlaSerLeuValAlaAlaAspGlyThrValTyrPheTyrLysGlu                              595600605                                                                     GTTAAGTCTGATTCAGCTAAGACAACCGGTACAGTAGTTGCAGGTACG2174                          ValLysSerAspSerAlaLysThrThrGlyThrValValAlaGlyThr                              610615620625                                                                  ACAACTGTTAAGTATGTTTACGAAAAAGCTGGTAGCGTTAATGTTAAC2222                          ThrThrValLysTyrValTyrGluLysAlaGlySerValAsnValAsn                              630635640                                                                     TTCGTTGACATCAATGGTAAAGTAATCAAAGCTCCTGTTTCAGATGAA2270                          PheValAspIleAsnGlyLysValIleLysAlaProValSerAspGlu                              645650655                                                                     AAAGATGCGAAACCTGGTTACAATTATGATACCGACTTGGATCAGAAA2318                          LysAspAlaLysProGlyTyrAsnTyrAspThrAspLeuAspGlnLys                              660665670                                                                     TTAGCTTCCATCACTTTTGAAGGCAAGGAATACAAACTTGTTCCTGCT2366                          LeuAlaSerIleThrPheGluGlyLysGluTyrLysLeuValProAla                              675680685                                                                     GGTGATTATCCGGTTGGTAAAGTTGGCAAGGGAAATAACTTGATTGAA2414                          GlyAspTyrProValGlyLysValGlyLysGlyAsnAsnLeuIleGlu                              690695700705                                                                  GTTGGTAATAATACTGCGAAAGGTATTGACCCAACAACAGGCAAAATT2462                          ValGlyAsnAsnThrAlaLysGlyIleAspProThrThrGlyLysIle                              710715720                                                                     GAAGCCGGTGTTAACAAAGAAGTTACCTATGTCTATAGAGCAGTGACA2510                          GluAlaGlyValAsnLysGluValThrTyrValTyrArgAlaValThr                              725730735                                                                     GGTTCTGTAGTTGTAAATTACAAAGATACAGAAGGTAATGTGATTAAA2558                          GlySerValValValAsnTyrLysAspThrGluGlyAsnValIleLys                              740745750                                                                     GATCCAGAAACGGATGTGTCTGATGCACCGGTTGGAGATGCTTATACT2606                          AspProGluThrAspValSerAspAlaProValGlyAspAlaTyrThr                              755760765                                                                     ACAACTGACAAGAAACCAAACGAAATCATCACAAAAGATGGATCACGC2654                          ThrThrAspLysLysProAsnGluIleIleThrLysAspGlySerArg                              770775780785                                                                  TATGTTCTTGTTCCATCTAAGACAGATGGTGAGGAAAATGGTAAAGTT2702                          TyrValLeuValProSerLysThrAspGlyGluGluAsnGlyLysVal                              790795800                                                                     ATCGAAGGAACAATCACAGTAACTTATGTTTACCAGAAAGTTGCAAAC2750                          IleGluGlyThrIleThrValThrTyrValTyrGlnLysValAlaAsn                              805810815                                                                     TGGATTCCAGAGATTCCAAATGTACCAGAAACAGACCGTCCAAAAGTA2798                          TrpIleProGluIleProAsnValProGluThrAspArgProLysVal                              820825830                                                                     CCTTACCCATTTGACCCAACAGAGCCAGACGAGCCAATCGATCCAACG2846                          ProTyrProPheAspProThrGluProAspGluProIleAspProThr                              835840845                                                                     ACACCAGGAACAAATGGCGAGGTTCCAAATATTCCTTACGTTCCAGGA2894                          ThrProGlyThrAsnGlyGluValProAsnIleProTyrValProGly                              850855860865                                                                  TATACACCGGTTGATCCTAAGGATAACACGCCGTTGAAACCAATTGAT2942                          TyrThrProValAspProLysAspAsnThrProLeuLysProIleAsp                              870875880                                                                     CCAAATGATCCAGGTAAGGGTTATGTACCACCAACACCAGAAAATCCA2990                          ProAsnAspProGlyLysGlyTyrValProProThrProGluAsnPro                              885890895                                                                     GGTGTTGATACACCAATTCCTTATGTTCCAGTTAAAAAAGTCGTAACT3038                          GlyValAspThrProIleProTyrValProValLysLysValValThr                              900905910                                                                     AACCACGTTGATGAAGAGGGTAACCCTATTGCACCGCAAGAAGAGGGA3086                          AsnHisValAspGluGluGlyAsnProIleAlaProGlnGluGluGly                              915920925                                                                     ACAAAACCAAACAAATCAATCCCAGGTTACGAGTTCACAGGTAAAACT3134                          ThrLysProAsnLysSerIleProGlyTyrGluPheThrGlyLysThr                              930935940945                                                                  GTTACTGACGAAGATGGCAACACAACTCACATCTACAAGAAAACACCA3182                          ValThrAspGluAspGlyAsnThrThrHisIleTyrLysLysThrPro                              950955960                                                                     GAAGTTAAGAATGGTACAGTTGTTGTTAACTATGTAACAGAAGATGGC3230                          GluValLysAsnGlyThrValValValAsnTyrValThrGluAspGly                              965970975                                                                     ACAGTTATCAAGGAACCTGTAACAGATACACCAACTTCTCCAGAAGGC3278                          ThrValIleLysGluProValThrAspThrProThrSerProGluGly                              980985990                                                                     ACACCATACGACACTACAGACAACAAACCTAAGACAATCACTTTCAAA3326                          ThrProTyrAspThrThrAspAsnLysProLysThrIleThrPheLys                              99510001005                                                                   GGTGAAGAGTATGAATTGGTTCGTGTTGACGGTACAGAAAACGGTAAA3374                          GlyGluGluTyrGluLeuValArgValAspGlyThrGluAsnGlyLys                              1010101510201025                                                              GTTGTAGAAGGTGAAACAGTTGTGACTTACGTTTACCGTAAAGTCGAA3422                          ValValGluGlyGluThrValValThrTyrValTyrArgLysValGlu                              103010351040                                                                  ACACCTGCTAAGAAAGTTGTAACTAACCACGTTGATGAAGAGGGTAAC3470                          ThrProAlaLysLysValValThrAsnHisValAspGluGluGlyAsn                              104510501055                                                                  CCTGTTGCGCCGCAAGAAGAGGGAACAAAACCAAACAAATCAATCCCA3518                          ProValAlaProGlnGluGluGlyThrLysProAsnLysSerIlePro                              106010651070                                                                  GGTTACGAATTTACAGGTAAAACTGTTACTGACGAAGATGGCAACACA3566                          GlyTyrGluPheThrGlyLysThrValThrAspGluAspGlyAsnThr                              107510801085                                                                  ACTCACATCTACAAGAAAACACCTGCTAAGAAAGTTGTGACTAACCAC3614                          ThrHisIleTyrLysLysThrProAlaLysLysValValThrAsnHis                              1090109511001105                                                              GTTGATGAAGAAGGTAACCCTATTGCTCCACAAGAGGATGGGACAACA3662                          ValAspGluGluGlyAsnProIleAlaProGlnGluAspGlyThrThr                              111011151120                                                                  CCAAAACGTCAAATTTCAGGTTACGAGTATGTGCGTACTGTAGTTGAT3710                          ProLysArgGlnIleSerGlyTyrGluTyrValArgThrValValAsp                              112511301135                                                                  GAAGAAGGTAACACGACACATATTTATCGCAAACTTTCTAATAAACCA3758                          GluGluGlyAsnThrThrHisIleTyrArgLysLeuSerAsnLysPro                              114011451150                                                                  ACAACACCTGAGAAGGAAACTCCTGCAAAACCTCAAGCAGGTAAAACC3806                          ThrThrProGluLysGluThrProAlaLysProGlnAlaGlyLysThr                              115511601165                                                                  GCTTCAGGTAAAGCTCAATTGCCAAATACTGGTGAGGCTTCATCTGTG3854                          AlaSerGlyLysAlaGlnLeuProAsnThrGlyGluAlaSerSerVal                              1170117511801185                                                              GCAGGTGCGCTTGGTACAGCAATGCTTGTCGCAACACTTGCGTTTGCA3902                          AlaGlyAlaLeuGlyThrAlaMetLeuValAlaThrLeuAlaPheAla                              119011951200                                                                  AGAAAACGTCGTCGTAACGAAGATTAGTCAAAATTCTTTATACAGAC3949                           ArgLysArgArgArgAsnGluAsp                                                      1205                                                                          TTTATTCCCCCACATAGAAAGTATAAGAATTGTACGTAACATGCAGGATTGCCTTTCCGA4009              AAAAATGAGGCTGGGCAAAAAGTCCAGAGTTACATCTTAGAGTTCGCTCCATTTCCAACC4069              TCCAACAGTCACTACTCTGACTGTTGGAGCTGTGTGGGGGTGGGAGACG4118                         __________________________________________________________________________

We claim:
 1. A DNA sequence comprising at least 15 contiguousnucleotides of(a) the nucleotide sequence set forth as SEQ ID NO: 1, 2or 3; or (b) a nucleotide sequence encoding the polypeptide set forth asSEQ ID NO: 1, 2 or
 3. 2. A DNA sequence according to claim 1 whichsequence contains at least 15 contiguous nucleotides selected fromnucleotides 2890-3306 of SEQ ID No
 2. 3. A DNA sequence according toclaim 1 containing at least 15 contiguous nucleotides selected fromnucleotides 1100-1934 of SEQ ID No
 3. 4. An expression sequence,comprising a DNA sequence according to claim 1 operatively linked to aregulating sequence for expression of the polypeptide encoded by saidDNA sequence.
 5. A polynucleotide probe for the diagnosis of aninfection by streptococcus suis, comprising a sequence according toclaim
 1. 6. A method for detecting an infection by a pathogenic strainof Streptococcus suis , comprising isolating DNA from a biologicalsample and contacting it with at least one probe according to claim 5,whereby the presence of the pathogenic strain of Streptococcus suis isdetected.
 7. A diagnostic kit for the detection of an infection by apathogenic strain of Streptococcus suis , wherein the kit contains atleast one probe according to claim
 5. 8. A plasmid or a viral vectorcomprising an expression sequence according to claim
 4. 9. A bacteriumtransformed with the plasmid or viral vector of claim 8.